UBRA.lV 


BACTERIOLOGY    AND    THE 
PUBLIC     HEALTH 


BACTERIOLOGY    AND 
THE  PUBLIC  HEALTH 


BY  GEORGE  NEWMAN,  M.D.,  F.E.S.K,  D.P.H. 

\N 

FORMERLY   DEMONSTRATOR   OF   BACTERIOLOGY   IN    KING'S   COLLEGE,    LONDON,    ETC. 

MEDICAL    OFFICER    OF.     HEALTH    OF    THE     METROPOLITAN    BOROUGH    OF   FINS1XURY 

JOINT-AUTHOR    OF   "  BACTERIOLOGY   OF   MILK." 


ILLUSTRATED 


THIRD  EDITION 


PHILADELPHIA 

P.   BLAKISTON'S   SON   AND   CO. 

1012  WALNUT  STREET 

1904 


N5 


BIOLOGY 

LIBRARY 

G 


Printed  in   Great  Britain. 

~~___^ 

^Ct:r  ^^^^* 


PREFACE 


THOUGH  nominally  a  third  edition  of  Bacteria  in  Relation  to  the 
Economy  of  Nature,  Industrial  Processes,  and  ttye  Public  Health,  this 
is,  speaking  generally,  a  new  book.  Several  new  chapters  have  been 
added,  and  the  whole  has  been  enlarged  and  revised. 

The  book  is  an  attempt  to  set  forth  a  simple  general  statement  of 
our  present  knowledge  of  bacteria,  especially  as  they  are  related  to  the 
public  health.  Theoretical  and  practical  text-books  of  bacteriology 
abound,  but  as  a  rule  they  deal  largely,  and  rightly  so,  with  laboratory 
methods  and  technique.  The  general  student  of  hygiene  and  the 
medical  officer  of  health  require,  however,  an  elementary  book  in 
which,  whilst  ample  laboratory  facts  are  recorded,  the  subject  is 
viewed  broadly  and  particularly  as  it  concerns  the  practical  everyday 
problems  of  health  and  preventive  medicine.  This  book  is  aimed  to 
meet  that  requirement. 

I  am  indebted  to  many  friends  and  colleagues  for  suggestions  and 
criticisms,  and  for  a  number  of  illustrations.  In  addition  to  a  number 
of  cliches  used  in  former  editions,  some  of  which  were  kindly  lent 
by  the  Scientific  Press,  Limited,  from  the  Atlas  of  Bacteriology, 
by  Slater  &  Spitta,  I  have  to  express  my  obligations  to  the 
Controller  of  His  Majesty's  Stationery  Office,  the  Secretary  of  the 
Eoyal  Commission  on  Sewage  Disposal,  and  the  Chairman  of  the 
Main  Drainage  Committee  of  the  London  County  Council,  for  permis- 
sion to  use  several  blocks  illustrating  sewage  bacteria  derived  from 
cultures  obtained  by  my  friend,  Dr  Houston,  in  the  course  of  his 
sewage  investigations.  I  am  in  a  similar  way  much  indebted  to  Mr 

vii 


o  f  \ 


viii  PREFACE 

Fouler  ton  of  the  Middlesex  Hospital,  and  Dr  Harold  Spitta  of  St 
George's  Hospital,  for  the  use  of  some  excellent  photographs.  My 
colleague,  Mr  Harold  Swithinbank,  has  kindly  allowed  me  to  use 
three  coloured  plates  of  "acid-fast"  cultures  from  our  book  on  the 
Bacteriology  of  Milk,  and  he  has  also  supplied  me  with  several  original 
plates.  To  each  of  these  gentlemen  I  am  glad  to  have  the  oppor- 
tunity of  expressing  my  sincere  thanks.  G.  N. 


LONDON,  August  1904. 


INTRODUCTION 


THE  science,  of  biology  has  for  its  object  the  study  of  organic  beings, 
and  for  its  end  the  knowledge  of  the  laws  of  their  growth, 
organisation,  and  function.  From  the  earliest  times  of  man  that  life 
has  been  studied  and  the  observations  recorded.  Thus  there  has  come 
slowly  to  be  a  considerable  accumulation  of  knowledge  concerning  the 
various  forms  (morphology)  and  functions  (physiology)  of  organised 
life.  In  the  midst  of  this  gradual  accumulation  of  facts  we  begin  to 
see  incoherence  becoming  coherent,  chaos  becoming  cosmos,  and 
apparent  chance  and  accident  becoming  law. 

Bacteriology  is  a  part,  a  chapter,  of  general  biology,  and  is 
concerned  with  the  facts,  as  at  present  known,  of  some  of  the  lowest 
forms  of  micro-organic  life.  Owing  to  a  variety  of  circumstances,  the 
chief  of  which  is  the  relation  of  these  micro-organisms  to  disease,  the 
study  of  bacteria  has  assumed  a  place  among  the  branches  of  biology 
of  somewhat  exceptional  importance.  The  application  of  biology  to 
daily  life  and  its  problems  has  in  recent  years  been  nowhere  more 
marked  than  in  the  realm  of  bacteriology,  where  the  great  names  of 
Pasteur,  Koch,  and  Lister,  represent  the  modern  epochs  of  advance. 
Turn  where  we  will,  we  shall  find  the  work  of  the  unseen  hosts  of 
bacteria  daily  claiming  more  and  more  attention  from  practical  people, 
and  thus  biology,  even  when  concerned  with  the  work  of  microscopic 
cells,  is  coming  to  occupy  a  new  place  in  the  minds  of  men.  Its 
evolution  begins  to  form  part  of  the  general  social  evolution. 

Certainly  the  recent  development  of  bacteriology  forms  a  remark- 
able feature  in  the  scientific  advance  of  our  time.  Not  only  in  the 
diagnosis  and  treatment  of  disease,  nor  even  in  the  various  applications 
of  preventive  medicine,  but  in  every  increasing  degree  and  sphere 
micro-organisms  are  recognised  as  agents  of  good  or  ill  no  longer  to 
be  ignored.  They  occur  in  our  drinking  water,  in  our  milk  supply,  in 
the  air  we  breathe.  They  ripen  cream,  and  flavour  butter.  They 
purify  sewage,  and  remove  waste  organic  products  from  the  land. 
They  are  the  active  agents  in  a  dozen  industrial  fermentations.  They 

«  (12 


x  INTRODUCTION 

assist  in  the  fixation  of  free  nitrogen,  and  they  build  up  assimilable 
compounds.  Their  activity  assumes  innumerable  phases  and  occupies 
many  spheres,  probably  more  frequently  proving  itself  beneficial 
than  injurious,  for  bacteria  are  both  economic  and  industrious  in  the 
best  sense  of  the  terms. 

Yet  bacteriology  has  its  limitations.  It  is  well  to  recognise  this, 
for  the  new  science  has  in  some  measure  suffered  in  the  past  from 
over-zealous  and  sanguine  friends.  It  cannot  achieve  everything 
demanded  of  it,  nor  can  it  furnish  a  causal  agent  for  every  disease  to 
which  human  flesh  is  liable.  It  is  a  science  which  even  yet  is  fuller 
of  hope  than  of  proved  and  established  knowledge,  for  we  are  at 
present  but  upon  the  threshold  of  the  matter.  As  in  the  neighbouring 
realm  of  chemistry,  it  is  to  be  feared  that  bacteriology  has  not  been 
without  its  alchemy.  The  interpretations  and  conclusions  which  have 
been  drawn  from  time  to  time  respecting  bacteriological  findings  have 
led  to  alarmist  or  optimist  views  which  have  not,  by  later 
investigations,  been  fully  confirmed.  For  the  science  has  had  devotees 
who  have  fondly  believed,  like  the  Alchemists,  that  the  twin  secret  of 
"transmuting  the  baser  metals  into  gold,"  and  of  indefinitely 
prolonging  human  life,  was  at  last  to  be  known.  Neither  the  worst 
fears  of  the  alarmist  nor  the  sanguine  hopes  of  the  optimist  have  been 
verified.  Science  does  not  progress  at  such  speed  or  with  such  kindly 
accommodation.  It  holds  many  things  in  its  hand,  but  not  finally  life 
or  death.  It  has  not  yet  brought  to  light  either  "  the  philosopher's 
stone  "  or  "  the  vital  essence." 

What  has  already  been  said  affords  ample  reason  for  a  wider 
dissemination  of  the  elementary  facts  of  bacteriological  science.  But 
there  are  other  reasons  of  a  more  practical  nature.  Municipalities 
and  other  bodies  are  expending  public  moneys  in  water  analysis,  in 
the  examination  of  milk  and  the  control  of  its  supply,  in  the  inspection 
of  cows  and  dairies,  in  the  bacterial  treatment  of  sewage,  in  pro- 
tecting the  oyster  trade,  in  the  ventilation  of  workshops  and  factories, 
in  disinfection,  in  the  prevention  of  epidemic  diseases,  and  in  other 
branches  of  public  health  administration.  Furthermore,  our  increasing 
colonial  possessions  with  their  tropical  diseases,  and  the  growth  of 
preventive  medicine  generally,  make  an  increasing  claim  upon  public 
opinion  and  those  engaged  in  raising  the  physical  condition  of  the 
people.  The  successful  accomplishment  and  solution  of  these 
questions  depends  in  measure  upon  a  correct  appreciation  of  the 
elements  of  bacteriology. 

The  present  is  a  transition  period  in  this  department  of  knowledge. 
A  very  large  body  of  facts  has  been  collected,  and  there  has  been  a 
natural  tendency  to  draw  somewhat  sweeping  deductions  which 
subsequent  knowledge  has  not  supported.  What  is  now  required  is 
that  our  experience  in  the  laboratory  and  outside  should  be  patiently 


INTRODUCTION  xi 

and  repeatedly  checked  and  tested.  If  the  science  of  bacteriology  is 
to  be  built  solidly,  the  two  necessities  of  accumulating  accurate  facts 
and  making  generalisations  and  deductions  must  proceed  side  by  side, 
the  former  being  well  established  before  the  latter  are  accepted.  It  is 
the  danger  of  a  new  science  that  too  much  is  expected  of  it. 
Bacteriology,  except  in  a  few  well-defined  spheres,  cannot  yet  stand 
alone  as  reliable  basis  for  legislation.  The  bacteriologist  must  be 
content  at  present  to  serve  as  indicator  rather  than  as  olictator.  The 
detection,  for  instance,  of  certain  bacteria  in  milk  or  in  oysters  is  an 
indication,  and  not  an  absolute  proposition,  of  unsatisfactory  dairying  or 
oyster  culture.  Common  sense  and  a  broad  view  of  all  the  ascertain- 
able  facts  must  guide  those  whose  business  it  is  to  apply  the  findings 
of  bacteriology  to  preventive  measures. 

In  the  pages  that  follow,  a  large  number  of  statements  occur  as  to 
the  external  circumstances  and  conditions  affecting  the  life  of  bacteria, 
and  to  understand  these  rightly  and  hold  them  in  right  proportion  to 
each  other,  it  is  necessary  to  bear  in  mind  that  many,  if  not  most  of 
them,  are  of  relative  importance.  They  are  of  value,  not  as  isolated 
units,  but  as  parts  of  a  whole.  It  is  their  co-ordination,  relativity, 
and  correlation  which  must  be  sought  after.  Again,  the  presence  of  a 
diphtheria  bacillus  in  the  throat  of  a  healthy  man  appears  at  first  sight 
to  be  a  fact  of  absolute  and  critical  importance  until  the  life-history  of 
the  bacillus  is  inquired  into  and  determined,  and  the  relation  of  the 
healthy  tissues  to  the  performance  of  its  function  understood.  The 
bacteriologist  and  worker  in  preventive  medicine  can  never  afford  to 
neglect  the  inter-relationship  which  exists  between  the  seed  and  the 
soil.  It  is  not  wholly  the  one  or  the  other  with  which  he  has  to  deal 
as  a  practical  man.  It  is  the  combination  and  the  inter-action 
between  the  two.  If  that  principle,  and  the  relativity  of  our  know- 
ledge of  bacteria  and  the  rdle  which  they  play  are  borne  in  mind, 
there  is  little  to  fear  from  a  transition  period. 

Whilst  there  can  then  be  no  doubt  as  to  the  advantage  of  a 
wide  dissemination  of  the  ascertained  facts  concerning  bacteria, 
especially  in  relation  to  water,  air,  milk,  and  other  foods,  it  must  not 
be  forgotten  that  only  patient  and  skilled  observation,  and 
experimental  research  in  well-equipped  laboratories,  can  advance  this 
branch  of  science  or  indeed  train  bacteriologists.  The  lives  of 
Darwin  and  Pasteur  adequately  illustrate  this  truth.  As  the  world 
learns  its  intimate  relation  to  science,  and  the  inter-dependence 
between  its  life  and  scientific  truth,  States  and  public  authorities  may 
be  expected  more  heartily  to  support  science. 


CONTENTS 

CHAPTEK  I 

THE  BIOLOGY  OF   BACTERIA 

PAGE 

Early  work — Place  of  Bacteria  in  Nature— Biology  of  Bacteria ;  Morphology, 
Composition,  Reproduction,  Influence  of  External  Conditions — Light 
—Modes  of  Bacterial  Action— Seed  and  Soil — Specificity  of  Bacteria 
— Association,  Antagonism,  Attenuation — Bacterial  Diseases  of  Plants  1-32 

CHAPTER  II 

BACTERIA    IN   WATER 

Quantity  of  Bacteria  in  Water— Quality  of  Water  Bacteria :  (a)  Ordinary 
Water  Bacteria ;  (6)  Sewage  Bacteria ;  B.  coli  communis ;  (c)  Patho- 
genic Bacteria  in  Water — Interpretation  of  the  Findings  of  Bacteri- 
ology—Natural Purification  of  Water— Artificial  Purification  of  Water 
—Sand  Filtration— Domestic  Purification  of  Water  .  .  .  33-72 


CHAPTER  III 

BACTERIA   IN   THE  AIR 

Methods  of  Examination  of  Air — Conditions  of  Bacterial  Contamination  of 
Air  :  (1)  Dust  and  Air  Pollution  ;  (2)  Moisture  or  Dampness  of  Surfaces : 
Bacteria  in  Sewer  Air ;  (3)  the  Influence  of  Gravity ;  (4)  Air  Currents. 
The  Relation  of  Bacteria  to  CO2  in  the  Atmosphere :  in  Workshops,  in 
Bakehouses,  in  Railway  Tubes,  in  the  House  of  Commons  .  .73-91 

xiii 


xiv  CONTENTS 

CHAPTEE  IV 

BACTERIA   AND   FERMENTATION 

PAGE 

Early  Work— Kinds  of  Fermentation  r  (1)  Alcoholic  Fermentation,  Asco- 
spores,  Pure  Cultures,  Films ;  (2)  Acetous  Fermentation ;  (3)  Lactic 
Acid  Fermentation  ;  (4)  Butyric  Fermentation ;  (5)  Ammoniacal  Fer- 
mentation— Diseases  of  Wine  and  Beer :  Turbidity,  Ropiness,  Bitter- 
ness, etc. — Industrial  Applications  of  Bacterial  Ferments  .  .  92-115 

CHAPTEK  V 

BACTERIA  IN   THE  SOIL 

Methods  of  Examination — Methods  of  Anaerobic  Culture— Place  and  Function 
of  Micro-organisms  in  Soil  —  Denitrification,  Nitrification,  Nitrogen- 
fixation,  Bacterial  Symbiosis — Saprophytic  and  Pathogenic  Organisms 
in  Soil— Tetanus— Quarter-Evil— Malignant  (Edema— The  Relation  of 
Soil  to  Bacterial  Diseases,  such  as  Typhoid  Fever  .  .  116-150 

CHAPTEE  VI 

THE  BACTERIOLOGY  OF  SEWAGE  AND  THE  BACTERIAL 
TREATMENT  OF  SEWAGE 

Composition  of  Sewage— Quantity  and  Quality  of  Bacteria  in  Sewage— Treat- 
ment of  Sewage :  (1)  Disposal  without  Purification  ;  (2)  Chemical  Treat- 
ment ;  (3)  Bacterial  Treatment — Evolution  of  Bacterial  Methods — Septic 
Tank  Method— Contact-Bed  Method— Manchester  Experiments— Effect 
of  Bacterial  Treatment  on  Pathogenic  Organisms  .  .  151-177 


CHAPTEE  VII 

BACTERIA   IN   MILK  AND   MILK   PRODUCTS 

General  Principles— Sources  of  Pollution— Number  of  Bacteria  in  Milk — 
Influence  of  Time  and  Temperature— Species  of  Bacteria  found  in  Milk 
— Fermentations  of  Milk — Pathogenic  Organisms  in  Milk— Milk-borne 
Disease:  Tuberculosis,  Typhoid  Fever,  Scarlet  Fever,  Sore-Throat 
Illnesses,  Cholera,  Epidemic  Diarrhoea— Preventive  Measures— Pro- 
tection of  Milk  Supply — Control  of  Milk  Supply :  Refrigeration,  Strain- 
ing, Sterilisation,  Pasteurisation — Specialised  Milk — Bacteria  in  Milk 
Products— Cream-Ripening — Butter-Making— Cheese-Making— Abnor- 
mal Cheese-Ripening— Poisonous  Cheese .  .  .  .  178-252 


CONTENTS  xv 

CHAPTEK  VIII 

BACTERIA  IN  OTHER   FOODS 

PAGE 

1.  Shell-fish,  Oysters,  Cockles,  Clams,  and  their  Relation  to  Disease; 
Symptoms  of  Oyster-borne  Disease ;  Channels  of  Infection ;  Preven- 
tive Methods — 2.  Meat  Poisoning ;  Tuberculous  Meat — 3.  Ice-cream 
and  Ice— 4.  Bacterial  Infection  of  Bread— 5.  Miscellaneous  Foods, 
Watercress,  etc.  .  .  .  .  »  .  .  253-279 

CHAPTEE  IX 

BACTERIA  AND   DISEASE 

Growth  of  Knowledge  of  Bacteria  as  Disease  Producers— Channels  of  Infec- 
tion— How  Bacteria  cause  Disease — Diphtheria:  Conditions  of  Infec- 
tion— Scarlet  Fever,  Typhoid  Fever,  Epidemic  Diarrhoea :  Conditions 
of  Infection— Suppuration  and  Abscess  Formation— Anthrax— Pneu- 
monia—Influenza— Actinomycosis— Glanders  .  .  .  280-324 


CHAPTEE  X 

TUBERCULOSIS   AS   A   TYPE  OF   BACTERIAL  DISEASE 

Pathology  and  Bacteriology  of  Tuberculosis — The  Bacillus  of  Koch — Animal 
Tuberculosis,  Bovine,  Avian,  etc. — Bovine  and  Human  Tubercle  Bacilli 
compared — Inter-communicability — Diagnosis  of  Bovine  Tubercle — The 
Prevention  of  Tuberculosis— Pseudo-Tuberculosis— Acid-fast  Bacteria 
Allied  to  the  Tubercle  Bacillus :  in  Man,  in  Animals,  in  Butter  and 
Milk,  in  Grass— Differential  Diagnosis— Streptothrix  Group  .  325-369 


CHAPTEE  XI 

THE   ETIOLOGY  OF  TROPICAL  DISEASES 

Malaria :  Forms  of  Malarial  Fever,  the  Mosquito  Theory,  Prevention  of 
Malaria — Cholera  :  Methods  of  Diagnosis — Plague  :  Symptoms,  Rats 
and  Plague,  Bacteriology,  Administrative  Considerations— Leprosy — 
Yellow  Fever— Malta  Fever— Sleeping  Sickness— Beri-beri  .  370-404 


xvi  CONTENTS 

CHAPTEK  XII 

THE  QUESTION    OF  IMMUNITY  AND  ANTITOXINS 

PAGE 

Bacterial  Products — Toxins — Question  of  Immunity — Kinds  of  Immunity — 
Theories  of  Immunity— Applications  of  Immunity — Vaccination  for 
Small-pox:  Effect  of  Vaccination — Pasteur's  Treatment  for  Rabies- 
Inoculations  for  Cholera,  Typhoid,  and  Plague— Antitoxin  Treatment 
of  Diphtheria  and  its  Effects  .  .  .  .  .  405-431 

CHAPTEE  XIII 

DISINFECTION 

General  Principles — Means  of  Disinfection :  by  Heat ;  by  Chemicals — 
Practical  Disinfection:  Rooms,  Walls,  Bedding,  Clothing,  Excreta, 
Books,  Linen,  Stables,  etc.— Disinfection  of  Hands— Disinfection  after 
Special  Diseases :  Phthisis,  Small-pox,  Scarlet  Fever,  Diphtheria, 
Typhoid,  Plague  .  .  .  .  .  .  .  432-451 


APPENDIX  ON  TECHNIQUE   ......     453-488 

INDEX  489-497 


LIST    OF    FIGURES 

FKJ.  PAGE 

1.  Various  Forms  of  Bacteria        .;             .  -.'-..  ,  .           7 

2.  Diagram  of  Sarcina  -   .              .   :          .              .  .  .8 

3.  Diagrams   of  Normal  and   Polymorphic   Forms  of  Tubercle 

Bacilli       .             .             .             .             .  .  .         10 

4.  Various  Forms  of  Spore  Formation  and  Flagella  .  .         13 

5.  Inoculating  Needles      .              .              .              .  .  .17 

6.  Media  for  Surface  and  Depth  Culture  .             .  .  ,17 

7.  Method    of   Producing  Hydrogen   by   Kipp's    Apparatus  for 

Cultivation  of  Anaerobes              .             .  .  .23 

8.  Koch's  Steam  Steriliser             .             .             .  .  .         24 

9.  Diagrams  of  B.  typhoms  and  B.  coli      .             .  .  .47 

10.  Pasteur-Chamberland  Filter      .             .             .  .  .71 

11.  Miquel's  Flask               .           ..             .             .  .  .74 

12.  Sedgwick's  Sugar-tube               .             .             .  .  .75 

13.  Diagram  of  Ascospore  Formation          .              .  .  .98 

14.  Gypsum  Block .             .             .             .             .  .  .98 

15.  Diagram  of  S.  ceremsice              .              .              .  .  .102 

16.  Diagram  of  S.  ellipsoidens          .             .             .  .  .102 

17.  Diagram  of  S.  pastorianus           .             .              .  .  .102 

18.  Frankel* s  Tube              ,             .             .             .  .  ,118 

19.  Rootlet  of  Pea  with  Nodules     .             .  .  .       133 

20.  Diagram  of  Bacillus  of  Symptomatic  Anthrax  .  .  .143 

21.  A  Plan  of  Septic  Tank  and  Filter-beds              .  .  .167 

22.  Contact-beds     ,  ,  ,  ....       169 

23.  "Ulax"  Filter  .             .             .             .             ,  .  .       229 

24.  Diagram  of  Bacillus  diphtherice  .              . .            .  .  .       289 

25.  Diagram  of  Types  of  Streptococci         .              .  .  .312 

xvij 


xviii  LIST  OF  FIGURES 

FIG.  PAGE 

26.  Diagram  of  Micrococcns  tetragonm-          .              .              .  .313 

27.  Diagram  of  Gonococcus  .....       314 

28.  Diagram  of  Bacillus  of  Anthrax  and  Blood  Corpuscles.  .       316 

29.  Quartan  Malaria  Parasite          .             .             .             .  .373 

30.  Tertian  Malaria  Parasite            .             .             .             .  .374 

31.  Malignant  Malaria  Parasite       .              .              .              .  .       374 

32.  Anopheles  maculipennis   .             .             .             .              .  .       377 

33.  Diagram  of  Culex  and  Anopheles         .             .             .  .       378 

34.  Human  and  Mosquito  Cycles  of  the  Malaria  Parasite  .  ' .       380 

35.  Diagram  of  the  Comma  Bacillus  of  Cholera      .             .  .       385 

36.  Suspended  Spinal  Cord              .             .             .             .  .421 

37.  Flask  used  for  Preparation  of  the  Toxin  of  Diphtheria  .       426 

38.  PetriDish           .             .             .            .             .           .,  .       453 

39.  A  Diagram  of  Colonies  of  Bacteria  on  a  Gelatine  Plate  .       454 

40.  The  Hanging  Drop       .             .             .             .             .  .455 

41.  Drying  Stage  for  Fixing  Films              .             .             .  .456 

42.  Types  of  Liquefaction  of  Gelatine         .             .             .  .       457 

43.  Levelling  Apparatus  for  Koch's  Plate  .             .             .  ^  ;    464 

44.  Moist  Chamber  for  Koch's  Plate           .             .             .  .464 

45.  Wolfhugel's  Counter     .             .             .             .             .  465 

46.  Filter-brushing  Method             .             .             .             .  .       466 

47.  Buchner  Tube  .             .-'.'.             .             .  .466 

48.  Another  Form  of  Buchner  Tube  478 


LIST    OF    PLATES 

[Note. — Photographs  marked  with  an  asterisk  (*)  have  been  kindly  lent  by  the 
Scientific  Press  Company;  those  marked  f  are  taken  by  permission  from  the 
Report  of  Royal  Commission  on  Sewage  Disposal;  and  those  marked  J  from 
Reports  to  the  London  County  Council.  ] 


PLATE 


1.  A  Form  of  Room  Temperature  Incubator    .  .   To  face  page    18 

2.  Hot-air  Steriliser  and  Blood-heat  Incubator             :.  „  24 

3.  B.  colt  communis  *;  Proteus  vulgaris  J              .              .  „  46 

4.  B.  coli  communis  ;  Gas  Production  in  Gelatine  f  „  50 

5.  Small  Centrifuge ;  Sedgwick's  Sugar-tube  „  74 

6.  Air-Plate  Culture  from  Labourer's  Cottage              .  „  76 

7.  Air- Plate  Cultures  from  Bakehouses  „  86 

8.  Saccharomyces   cerevisice ;    Ascospores ;    Pathogenic 

Yeast  ..*...             ,  „  98 

9.  Buchner's  Tube  ;•  Kipp's  Apparatus  for  Anaerobic 

Culture             .             .                           .             .  „  116 

10.  A  Vacuum  Method  of  Anaerobic  Culture    .             .  „  118 

11.  Nitrous   Organism;    Nitric   Organism;    Nitrogen- 

fixing  Organisms          .              .              .              .  „  128 

12.  Nitrogen-fixing    Bacteria    in    Nodule    on    Rootlet 

of  Pea              .             •'•'•••             •             •  «  134 

13.  B.  tetani \*;  B.  mycoides*;  Streptothrix  actinomyces  ; 

B.  mallei.             .             .             .             .             .  „  140 

14.  Sewage  Proteus,  Organism  and  Plate  Culture  J       .  „  154 

15.  Sewage  Streptococci  t  and  Streptococcus  pyogenes  *  .  „  156 

16.  B.  mesentericus,  Organism  and  Plate  Culture  J          .  „  158 

17.  B.    anthracis,  from    Septic    Tank    Liquor   and    in 

Gelatine  Culture  (impression)  f            .              .  „  176 

18.  B.  tuberculosis  (old  culture);    Tubercle  Bacilli   in 

Cow's  Udder   .  204 


xx  LIST  OF  PLATES 

PLATE 

19.  B.  diphtheric?;  B.  von  II of  maim        .  .  .   To  face  page  288 

20.  B.typhosus;  B.  typhosus  (flagella)*;  Widal-Griiber 

Reaction  *  ;  B.  typhosus  in  Human  Mesenteric 

Gland.  .  .  302 

21.  B.  enteritidis  sporogenes\;  "Enteritidis  change"  in 

Milk  Cultures!  „  307 

22.  B.    anthracis   (Stab    Culture)  f;    B.    anthracis  from 

Blood*;  Frankel' s  Pneumococcus      .  ,  „  318 

23.  B.  tuberculosis,  from  Sputum,*  Tissues,  and  Culture  „  328 

24.  Comparative  Cultures  of  Tubercle  Bacillus  (Bird, 

Mammal,  Butter)  „  350 

25.  Cultures  of  Butter  Bacillus  of  Rabinowitsch  and 

Mailer's  Milk  Bacillus  (Chromo)      .  .  „  360 

26.  Cultures  of  B.  friburgemis,  Nos.  I.  and  II.  (Chromo)  „  362 

27.  Cultures  of  Butter  Bacilli  of  Binot  and  Grassberger 

(Chromo)          ......  364 

28.  Comparative  Cultures  of  Acid- fast  Bacteria  (Grass 

and  Manure)    ......  366 

29.  Streptothrix  luteola  (Foulerton) ;  Streptothrix  hominis 

(Foulerton)      ....  ,,368 

30.  B.  leprce  ;  B.  pestis*;  Staphylococcus  pyogenes  aureus  t,  398 

31.  Apparatus  for    Filter-brushing   Method  in  ^Water 

Examination  466 


OF  THE 

UNIVERSITY 


BACTERIOLOGY    AND    PUBLIC 
/       HEALTH 

CHAPTEE  I 

THE  BIOLOGY  OF   BACTERIA* 

Early  work  —  Place  of  Bacteria  in  Nature  —  Biology  of  Bacteria;  Morphology, 
Composition,  Reproduction,  Influence  of  External  Conditions  —  Light  — 
Modes  of  Bacterial  Action  —  Seed  and  Soil  —  Specificity  of  Bacteria  — 
Association,  Antagonism,  Attenuation  —  Bacterial  Diseases  of  Plants. 

THE  first  scientist  who  demonstrated  the  existence  of  micro-organisms 
was  Antony  von  Leeuwenhoek.  He  was  born  at  Delft,  in  Holland, 
in  1632,  and  enthusiastically  pursued  microscopy  with  primitive 
instruments.  He  corroborated  Harvey's  discovery  of  the  circulation 
of  the  blood,  in  the  web  of  a  frog's  foot  ;  he  defined  the  red  blood 
corpuscles  of  vertebrates,  the  fibres  of  the  lens  of  the  human  eye, 
the  scales  of  the  skin,  and  the  structure  of  hair.  He  was  neither 
educated  nor  trained  in  science,  but  in  the  leisure  time  of  his 
occupation  as  a  linen-draper  he  learned  the  art  of  grinding  lenses, 
in  which  he  became  so  proficient  that  he  was  able  to  construct  a 
microscope  of  greater  power  than  had  been  previously  manufactured. 
The  compound  microscope  dates  from  1590,  and  when  Leeuwenhoek 

*  We  propose  throughout  to  use  the  term  bacterium  (pi.  bacteria)  in  its  generic 
meaning,  unless  especially  stated  to  the  contrary.  It  will  also  be  synonymous  with 
the  terms  microbe,  germ,  and  micro-organism.  The  term  bacillus  will,  of  course,  be 
restricted  to  a  rod-shaped  bacterium. 

A 


2  THE  BIOLOGY  OF  BACTERIA 

was  about  forty  years  old,  Holland  had  already  given  to  the  world 
both  microscope  and  telescope.  Eobert  Hooke  did  for  England 
what  Hans  Janssen  had  done  for  Holland,  and  established  the  same 
conclusion  that  Leeuwenhoek  arrived  at  independently,  viz.,  that  a 
simple  globule  of  glass  mounted  between  two  metal  plates  which 
were  pierced  with  a  minute  aperture  to  allow  rays  of  light  to  pass 
was  a  contrivance  which  would  magnify  more  highly  than  the 
recognised  microscopes  of  that  day.  It  was  with  some  such  instru- 
ment as  this  that  the  first  micro-organisms  were  observed  in  a  drop 
of  water.  It  was  not  until  more  than  a  hundred  years  later  that 
these  "  animalcula,"  as  they  were  termed,  were  thought  to  be  anything 
more  than  accidental  to  any  fluid  or  substance  containing  them. 
Plenciz,  of  Vienna,  was  one  of  the  first  to  conceive  the  idea  that 
decomposition  could  only  take  place  in  the  presence  of  some  of  these 
"animalcula."  This  was  in  the  middle  of  the  eighteenth  century. 
Just  about  a  century  later,  by  a  series  of  important  discoveries,  it 
was  established  beyond  dispute  that  these  micro-organisms  had  an 
intimate  causal  relation  to  fermentation,  putrefaction,  and  disease. 
Spallanzani,  Pasteur,  and  Tyndall  are  the  three  workers  who  more 
than  others  contributed  to  this  discovery.  Spallanzani  was  an  Italian 
who  studied  at  Bologna,  and  was  in  1*754  appointed  to  the  Chair  of 
Logic  at  Eeggio.  But  his  inclinations  led  him  into  the  realm  of 
natural  history.  Amongst  other  things,  his  attention  was  directed 
to  the  doctrine  of  spontaneous  generation,  which  had  been  propounded 
by  Needham  a  few  years  previously.  In  1768  Spallanzani  became 
Professor  of  Natural  History  at  Pavia,  and  whilst  there  he  demon- 
strated that  if  infusions  of  vegetable  matter  were  placed  in  flasks 
and  hermetically  sealed,  and  then  brought  to  the  boiling  point,  no 
living  organisms  could  thereafter  be  detected,  nor  did  the  vegetable 
matter  decompose.  When,  however,  the  flasks  were  but  slightly 
cracked,  the  air  gained  admittance,  then  invariably  both  organisms 
and  decomposition  appeared.  Schwann,  the  founder  of  the  cell- 
theory,  and  Schultze,  both  showed  that  if  the  air  gaining  access  to 
the  flask  were  either  calcined  or  drawn  through  strong  acid  the 
result  was  the  same  as  if  no  air  entered  at  all,  namely,  there  were  no 
organisms  and  there  was  no  decomposition.  The  result  of  these  investi- 
gations was  that  scientific  men  began  to  believe  that  no  form  of 
life  arose  de  novo  (aliogenesis),  but  had  its  source  in  previous  life 
(biogenesis).  It  remained  for  Pasteur  and  Tyndall  to  demonstrate 
this  beyond  dispute,  and  to  put  to  rout  the  fresh  arguments  for 
spontaneous  generation  which  Pouchet  had  advanced  as  late  as  1859. 
Pasteur  collected  the  floating  dust  of  the  air,  and  found  by  means 
of  the  microscope  many  organised  particles,  which  he  sowed  on 
suitable  infusions,  and  thus  obtained  rich  crops  of  "animalcula." 
He  also  demonstrated  that  these  organisms  existed  in  varying 


SPONTANEOUS  GENERATION  3 

degrees  in  different  atmospheres,  few  in  the  pure  air  of  the  Mer  de 
Glace,  more  in  the  air  of  the  plains,  most  in  the  air  of  towns.  He 
further  proved  that  it  was  not  necessary  to  insist  upon  hermetic 
sealing  or  cotton  filters  to  keep  these  living  organisms  in  the 
air  from  gaining  access  to  a  flask  of  infusion.  If  the  neck  of  the 
flask  were  drawn  out  into  a  long  tube  and  turned  downwards,  and 
then  a  little  upwards,  even  though  the  end  be  left  open,  no  con- 
tamination gained  access.  Hence,  if  the  infusion  were  boiled,  no 
putrefaction  would  occur.  The  organisms  which  fell  into  the  open 
end  of  the  tube  were  arrested  in  the  condensation  water  in  the 
angle  of  the  tube ;  but  even  if  that  were  not  so,  the  force  of  gravity 
acting  upon  them  prevented  them  from  passing  up  the  long  arm  of 
the  tube  into  the  neck  of  the  flask.  A  few  years  after  Pasteur's 
first  work  on  this  subject,  Tyndall  conceived  a  precise  method  of 
determining  the  absence  or  presence  of  dust  particles  in  the  air  by 
passing  a  beam  of  sunlight  through  a  glass  box  before  and  after  its 
walls  had  been  coated  with  glycerine.  Into  the  floor  of  the  box 
were  fixed  the  mouths  of  flasks  containing  an  infusion.  These 
were  boiled,  after  which  they  were  allowed  to  cool,  and  might 
then  be  kept  for  weeks  or  months  without  putrefying  or  reveal- 
ing the  presence  of  germ  life.  Here  all  the  conditions  of  the  in- 
fusions were  natural,  except  that  in  the  air  above  them  there  was  no 
dust. 

The  sum-total  of  result  arising  from  these  investigations  was  to 
the  effect  that  no  spontaneous  generation  was  possible,  that  the 
atmosphere  contained  unseen  germs  of  life,  that  the  smallest  of 
organisms  responded  to  the  law  of  gravitation  and  adhered  to  moist 
surfaces,  and  that  micro-organisms  were  in  some  way  or  other  the 
cause  of  putrefaction. 

The  final  refutation  of  the  hypothesis  of  spontaneous  generation 
was  followed  by  an  awakened  interest  in  the  unseen  world  of  micro- 
organic  life.  Investigations  into  fermentation  and  putrefaction 
followed  each  other  rapidly,  and  in  1863  Davaine  claimed  that 
Pollender's  bacillus  of  anthrax,  which  was  found  in  the  blood  and 
tissues  of  animals  which  had  died  of  anthrax,  was  the  cause  of  that 
disease.  From  that  time  to  this,  in  every  department  of  biology, 
bacteria  have  been  increasingly  found  to  play  an  important  part. 
They  cause  changes  in  milk,  and  flavour  butter;  they  decompose 
animal  matter,  yet  build  up  the  broken-down  elements  into  com- 
pounds suitable  for  use  in  nature's  economy;  they  assist  in  the 
fixation  of  free  nitrogen ;  they  purify  sewage ;  in  certain  well- 
established  cases  they  are  the  cause  of  specific  disease,  and  in  many 
other  cases  they  are  the  probable  cause.  No  doubt  the  disposal  of 
spontaneous  generation  did  much  to  arouse  interest  in  this  branch 
of  science.  Yet  it  must  not  be  forgotten  that  the  advance  of  the 


4  THE  BIOLOGY  OF  BACTERIA 

microscope  and  bacteriological  method  and  technique  have  played  a 
large  share  in  this  development.  The  sterilisation  of  culture  fluids 
by  heat,  the  use  of  aniline  dyes  as  staining  agents,  the  introduction 
of  solid  culture  media  (such  as  gelatine  and  agar),  and  Koch's 
"  plate "  method,  have  all  contributed  not  a  little  to  the  enormous 
advance  of  bacteriology. 


The  Place  of  Bacteria  in  Nature 

As  we  have  seen,  for  a  considerable  period  of  time  after  their  first 
detection  these  unicellular  organisms  were  considered  to  be  members 
of  the  animal  kingdom.  As  late  as  1838,  when  Ehrenberg  and 
Dujardin  drew  up  their  classification,  bacteria  were  placed  among  the 
Infusorians.  This  was  in  part  due  to  the  powers  of  motion  which 
these  observers  detected  in  bacteria.  It  is  now,  of  course,  recognised 
that  animals  have  no  monopoly  of  motion.  But  what,  after  all, 
are  the  differences  between  animals  and  vegetables  so  low  down  in 
the  scale  of  life  ?  Chiefly  two :  there  is  a  difference  in  life-history 
(in  structure  and  development),  and  there  is  a  difference  in  pabulum. 
A  plant  secures  its  nourishment  from  much  simpler  elements  than  is 
the  case  with  animals ;  for  example,  it  obtains  its  carbon  from  the 
carbonic  acid  gas  in  air  and  water.  This  it  is  able  to  do,  as  regards 
the  carbon,  by  means  of  the  green  colouring  matter  known  as  chloro- 
phyll, by  the  aid  of  which,  with  sunlight,  carbonic  acid  is  decomposed 
in  the  chlorophyll  corpuscles,  the  oxygen  passing  back  into  the 
atmosphere,  the  carbon  being  stored  in  the  plant  in  the  form  of 
starch  or  other  organic  compound.  The  supply  of  carbon  in  the 
chlorophyll-free  plants,  amongst  which  are  the  bacteria,  is  obtained 
by  breaking  up  different  forms  of  carbohydrates.  Beside  albumen 
and  peptone,  they  use  sugar  and  similar  carbohydrates  and  glycerine 
as  a  source  of  carbon.  Many  of  them  also  have  the  capacity  of  using 
organic  matters  of  complex  constitution  by  converting  such  into 
water,  carbonic  acid  gas,  and  ammonia.  Their  hydrogen  comes  from 
water,  their  nitrogen  from  the  soil,  chiefly  in  the  form  of  nitrates. 
From  the  soil,  too,  they  obtain  other  necessary  salts.  Now  all  these 
substances  are  in  elementary  conditions,  and  as  such  plants  can 
absorb  them.  Animals,  on  the  other  hand,  are  only  able  to  utilise 
compound  food  products  which  have  been,  so  to  speak,  prepared  for 
them,  for  example  albuminoids  and  proteids.  They  cannot  directly 
feed  upon  the  elementary  substances  forming  the  diet  of  vegetables. 
This  distinction,  however,  did  not  at  once  clear  up  the  difficult 
matter  of  the  classification  of  bacteria.  It  is  true,  they  possess  powers 
of  motion,  are  free  from  chlorophyll,  and  even  feed  occasionally  upon 
products  of  decomposition — three  physiological  characters  which 


UNIVERSITY 

OF 


CLASSIFICATION  OF 

would  ally  them  to  the  animal  kingdom.  Yet  by  their  structure  and 
capsule  of  cellulose  and  by  their  life-history  and  mode  of  growth 
they  unmistakably  proclaim  themselves  to  be  of  the  vegetable 
kingdom.  In  1853  Cohn  arrived  at  a  conclusion  to  this  effect,  and 
since  that  date  bacteria  have  become  more  and  more  limited  in  clas- 
sification and  restricted  in  definition. 

Even  yet,  however,  we  are  far  from  a  scientific  classification  of 
bacteria.  Nor  is  this  matter  for  surprise.  The  development  in  this 
branch  of  biology  has  been  so  rapid  that  it  has  been  impossible  to 
assimilate  the  facts  collected.  The  facts  themselves  by  their 
remarkable  variety  have  not  aided  classification.  Names  which  a  few 
years  ago  were  applied  to  individual  species,  like  Bacillus  subtilis,  or 
Bacterium  termo,  or  Bacillus  coli,  are  now  representative,  not  of 
individuals,  but  of  families  and  species.  Again,  isolated  character- 
istics of  certain  microbes  such  as  motility,  power  of  liquefying 
gelatine,  size,  colour,  and  so  forth,  which  at  first  sight  might  appear 
as  likely  to  form  a  basis  for  classification,  are  found  to  vary  not  only 
between  similar  germs,  but  in  the  same  germ.  Different  physical 
conditions  have  so  powerful  an  influence  upon  these  microscopic  cells 
that  their  individual  characters  are  constantly  undergoing  change. 
For  example,  bacteria  in  old  cultures  assume  a  different  size,  and 
often  a  different  shape,  from  younger  members  of  precisely  the  same 
species;  Bacillus  pyocyaneus  produces  a  green  to  olive  colour  on 
gelatine,  but  a  brown  colour  on  potato ;  the  bacillus  of  Tetanus  is 
virulently  pathogenic,  and  yet  may  not  act  thus  unless  in  com- 
pany with  certain  other  micro-organisms.  Hence  it  will  at  once 
appear  to  the  student  of  bacteriology  that,  though  there  is  great 
need  for  classification  amongst  the  six  or  seven  hundred  named 
"species"  of  microbes,  our  present  knowledge  of  their  life-history 
is  not  yet  advanced  enough  to  form  more  than  a  provisional 
arrangement. 

We  know  that  bacteria  are  allied  to  Hyphomycetes  on  the  one 
hand  and  Saccharomycetes  on  the  other,  and  that  they  have  no 
differentiation  into  root,  stem,  or  leaf ;  we  know  that  they  are  fungi 
(having  no  chlorophyll),  in  which  no  sexual  reproduction  occurs,  and 
that  their  mode  of  multiplication  is  by  division.  From  such  facts  as 
these  we  may  build  up  a  classification  as  follows : — 


[VEGETABLE  KINGDOM. 


THE  BIOLOGY  OF  BACTERIA 


VEGETABLE    KINGDOM. 


Thallophyta. 

[  =  The  lowest  forms 
of  vegetable  life.  No 
differentiation  into 
root,  stem,  or  leaf.] 
I 


Muscineae. 


I  I 

Pteridophyta.  Phanerogam  ia. 


Algae. 

[= Chlorophyll 
present.] 


Fungi. 

[  =  No  Chlorophyll.] 


Hymenomycetes.    Hyphomycetes.    Blastoraycetes.        Schizomycetes 
(Mushrooms,  etc.)  (Moulds.)  (Yeasts,  etc.)       [  =  multiplication  by  cell 

division  or  by  spores] 

or 
Bacteria. 


Ml)  Coccacese  ;;  —  r( 
cells. 

(2)  Bacteriacese  — 

and  threads. 

(3)  Leptotrichese. 

(4)  Cladotrichese. 


*  Migula  has   suggested  that  the  Schizomycetes  should   be   subdivided  into  Coccacece,  Bacteriacecv,  Spirillac 
(spirilla,  spirochreta),  Chlamydobacteriacece  (Streptothrix,  Crenothrix,  Cladothrix),  and  Ikggiatoa. 


Morphology:  Structure  and  Form 

Having  now  located  micro-organisms  in  the  economy  of  nature, 
we  may  proceed  to  describe  their  subdivisions  and  form.  For 
practical  convenience  rather  than  theoretical  accuracy,  we  may  accept 
the  simple  division  of  the  family  of  bacteria  into  three  chief  forms, 
viz. : — 

(  (1)  Round  cell  form — coccus. 
Lower  Bacteria-!    (2)  Eod  form — 'bacillus. 

(  (3)  Thread  form — spirillum. 
Higher  Bacteria — Leptothrix,  Streptothrix,  Cladothrix,  etc. 

A  classification  dependent  as  this  is  upon  the  form  alone  is  not  by 
any  means  ideal,  for  it  ignores  all  the  complicated  functions  of 
bacteria,  but  it  is,  as  we  have  said,  practically  convenient. 

1.  The  Coccus. — This  is  the  group  of  round  cells.  They  vary  in 
size  as  regards  species,  and  as  regards  the  conditions,  artificial  or 
natural,  under  which  they  have  been  grown.  Some  are  less  than 
2- WOTF  °f  an  incn  m  diameter ;  others  are  half  as  large  again,  if  the 
word  large  may  be  used  to  describe  such  minute  objects.  No  regular 
standard  can  be  laid  down  as  reliable  with  regard  to  their  size. 
Hence  the  subdivisions  of  the  cocci  are  dependent  not  upon  the 
individual  elements  so  much  as  upon  the  relation  of  those  elements  to 
each  other.  A  simple  round  cell  of  approximately  the  size  already 
named  is  termed  a  microcowus  (jumcpos,  small).  Certain  species  of 


FORMS  OF  BACTERIA 


inicrococci  always  or  almost  always  occur  in  pairs,  and  such  a  com- 
bination is  termed  a  diplococcus.  Some  diplococci  are  united  by  a 
thin  capsule,  which  may  be  made  apparent  by  special  methods  of 
staining;  in  others  no  limiting  or  uniting  membrane  can  be  seen 
with  the  ordinary  high  powers  of  the  microscope.  Again,  one  fre- 
quently finds  a  species  which  is  exactly  described  by  saying  that  two 
inicrococci  are  in  contact  with  each  other,  and  move  and  act  as  one 
individual,  but  otherwise  show  no  alteration ;  whilst  others  are  seen 
which  show  a  flattening  of  the  side  of  each  micrococcus  which  Is  in 
relation  to  its  partner.  Perhaps  the  diplococci  in  an  even  greater 
degree  than  the  micro- 
cocci  respond  to  external 
conditions  both  as  regards 
size  and  shape.  It  must 
further  be  borne  in  mind 
that  a  dividing  micrococcus 
assumes  the  exact  appear- 
ance of  a  diplococcus 
during  the  transition  stage 
of  the  fission.  Hence,  with 
the  exception  of  several 
well  -  marked  species  of 
diplococci,  this  form  is 
somewhat  arbitrary.  The 
third  kind  of  micrococcus  is 
that  formed  by  a  number 
of  elements  in  a  twisted 
chain,  named  streptococcus 
(orrpeTTTo?,  twisted).  This 
form  is  produced  by  cells 
dividing  in  one  axis,  and 
remaining  in  contact  with 
each  other.  It  occurs  in  a 
number  of  different  species, 
or  what  are  supposed  by  many  authorities  to  be  different  species, 
owing  to  their  different  effects.  Morphologically  all  the  streptococci 
are  similar,  though  a  somewhat  abortive  attempt  has  been  made  to 
divide  them  into  two  groups,  according  as  to  whether  they  were  long 
chains  or  short.  As  a  matter  of  fact,  the  length  of  streptococci 
depends  in  some  cases  upon  biological  properties,  in  others  upon 
external  treatment  or  the  medium  of  cultivation  which  has  been  used. 
Sometimes  they  occur  as  straight  chains  of  only  half  a  dozen 
elements ;  at  other  times  they  may  contain  thirty  or  forty  elements, 
and  twist  in  various  ways,  even  forming  rosaries.  The  elements,  too, 
differ  not  only  in  size,  but  in  shape,  appearing  occasionally  as  oval 


FIG.  1.— DIAGRAMS  OF  VARIOUS  FORMS  OF  BACTERIA. 


1.  Micrococcus. 

2.  Diplococcus. 

3.  Streptococcus. 


4.  Staphylococcus.  7.  Sarcina. 

5.  Leuconostoc,  show-  8    Bacillus. 

ing  Arthrospores.  9.  Spirillum. 

6.  Merismopedia. 


8  THE  BIOLOGY  OF  BACTERIA 

cells  united  to  each  other  at  their  sides.  The  fourth  form  is  consti- 
tuted by  the  micrococci  being  arranged  in  masses  like  grapes,  the 
staphylococcus  ((rTa<j>v\i?,  a  bunch  of  grapes).  The  elements  are 
often  smaller  than  in  the  streptococcus,  and  the  name  itself  describes 
the  arrangement.  There  is  no  matrix  and  no  capsule.  This  is  the 
commonest  organism  found  in  abscesses,  etc.  The  sarciiia  is  best 

classified  amongst  the  cocci,  for  it  is 
composed  of  them,  in  packets  of  four 
or  multiples  of  four,  produced  by  divi- 
sion vertically  in  two  planes.  If  the 
division  occurs  in  one  plane,  we  have 
as  a  result  small  squares  of  round  cells 
known  as  merismopedia.  In  both  these 
conditions  it  frequently  happens  that 
the  contiguous  sides  of  the  elements  of 
packets  become  faceted  or  straightened 
against  each  other.  It  may  happen, 
too,  particularly  in  the  sarcincc,  that 
FIG.  2,-Diagram  of  sarcina.  segmentation  is  not  complete,  and  that 

the   elements   are   larger  than  in  any 

other  class  of  cocci.  They  stain  very  readily.  Nearly  all  the  cocci 
are  non-motile,  though  Brownian  movement  (see  p.  11)  may  readily 
be  observed. 

2.  The  Bacillus. — This  group   consists   of  rods,   having  parallel 
sides  and  being  longer  than  they  are  broad.     They  differ  in  every 
other   respect  according  to   species,  but   these   two   characteristics 
remain  to  distinguish  them.     Many  of  them  are  motile,  others  not. 
The  ends  or  poles  of  a  bacillus  may  be  pointed,  round,  or  almost 
exactly   square  and   blocked.      They  all,  or   nearly   all,  possess   a 
capsule.      Individuals    of    the    same    species    may   differ    greatly, 
according  to  whether  they  have  been  naturally  or  artificially  grown, 
and  pleomorphic  forms  are  abundant. 

3.  The  Spirillum. — This  wavy-thread  group  is  divisible  into  a 
number  of  different  forms,  to  which  authorities  have  given  special 
names.      It   is  sufficient,  however,  to  state  that  the  two  common 
forms  are  the  non-septate  spiral  thread  (e.g.  the  Spirillum  Obermeier 
of  relapsing  fever),  which  takes  no   other  form  but  a  lengthened 
spirillum ;  and  the  spirillum  which  breaks  up  into  elements  or  units, 
each  of  which  appears  comma-shaped  (e.g.  the  cholera  bacillus).      The 
degree  of  curvature  in  the  spirilla,  of  course,  varies.     They  are  the 
least  important  of  the  lower  bacteria. 

The  Higher  Bacteria  group  includes  more  highly  organised 
members  of  the  Schizomycetes.  They  possess  filaments,  which  may 
be  branched,  and  almost  always  have  septa  and  a  sheath.  Perhaps 
the  most  marked  difference  from  the  lower  bacteria  is  in  their 


POLYMORPHISM  9 

reproduction.  In  the  higher  bacteria  we  may  have  what  is  in  fact 
a  flower — terminal  fructification  by  conidia.  In  this  group  of 
vegetables  we  have  the  Beggiatoa,  Leptothrix,  Cladothrix,  and,  at  the 
top,  the  Streptothrix.  It  has  been  demonstrated  that  Strcptothrix 
actinomycotica  and  Streptothrix  madurce  are  the  organismal  cause, 
respectively,  of  Actinomycosis  and  Madura-foot,  two  diseases  which 
had  hitherto  been  obscure. 

Polymorphism  (or  Pleomorphism). — This  term  is  used  to  designate 
an  inconstancy  of  form  or  a  tendency  towards  biological  variation. 
Vibrios  may  become  spirilla,  the  ray  fungus  passes  through  a  coccoid 
and  bacillary  stage,  and  the  diphtheria  bacillus  may  either  be  long, 
short,  straight,  or  clubbed.  This  diversity  of  form  appears  to  belong 
to  many  species,  and  is  transmitted  from  generation  to  generation ; 
or  the  various  forms  may  occur  in  succession,  and  represent  different 
stages  in  the  life-history.  In  B.  diphtherice,  B.  pestis,  and  B.  tuber- 
culosis and  other  forms,  polymorphism  undoubtedly  occurs.  It 
is  particularly  marked  in  very  old  cultures  of  the  last  named. 
The  ordinary  well-known  bacillus  may  grow  out  into  threads  with 
bulbous  endings,  granular  filaments,  "drumsticks,"  and  diplococcal 
forms.  It  is  now  known  that  amongst  the  causes  of  polymorphism 
are  certain  adverse  conditions  of  medium  or  other  physical  influences 
(moisture,  temperature,  age,  etc.),  and  thus  some  bacteria,  especially 
bacilli  or  vibrios,  become  altered  in  shape,  losing  their  ordinary  form. 
On  transferring  such  aberrant  and  abnormal  forms  to  fresh  medium 
or  favourable  conditions,  they  are  generally  able  to  assume  their 
original  morphology.  Indeed  the  aberrant  form  is  in  all  probability 
only  a  stage  in  their  life-history.  Involution  forms  usually  imply 
degeneration. 

Biology  of  Bacteria 

Composition. — From  what  we  have  seen  of  the  pabulum  of 
micro-organisms,  we  should  conclude  that  in  some  form  or  other  they 
contain  the  elements  nitrogen,  carbon,  and  hydrogen.  All  three 
substances  are  combined  in  the  mycoprotein  or  protoplasm  of  which 
the  body  of  the  microbe  consists.  This  is  generally  homogeneous, 
proteid  material,  and  there  is  no  sign  of  a  nucleus.  It  possesses  a 
marked  affinity  for  aniline  dyes,  and  by  this  means  organisms  are 
stained  for  the  microscope.  Besides  the  variable  quantity  of 
nitrogen  present,  mycoprotein  may  also  contain  various  mineral 
salts.  The  uniformity  of  the  cell-protoplasm  may  be  materially 
affected  by  disintegration  and  segmentation  due  to  degenerative 
changes.  Vacuoles,  which  it  is  necessary  to  differentiate  from  spores, 
also  may  appear  from  a  like  cause.  Vacuolation  may  also  occur 
as  a  result  of  a  process  of  osmosis  in  salt  solutions,  the  protoplasm 
of  the  bacillus  becoming  contracted  and  disintegrated  (plasmolysis). 


10  THE  BIOLOGY  OF  BACTERIA 

Two  other  signs  of  degeneration  are  the  appearance  of  granules  in 
the  body  of  the  cell-protoplasm  known  as  metachromatic  granules, 
owing  to  their  different  staining  propensities,  and  the  polar  bodies 
which  are  seen  in  some  species  of  bacteria.  Surrounding  the  mass 
of  mycoprotein,  we  find  in  most  organisms  a  capsule  or  membrane 
composed,  in  part  at  least,  of  cellulose.  This  sheath  plays  a  protective 
part  in  several  ways.  During  the  adult  stage  of  life  it  protects  the 
mycoprotein,  and  holds  it  together.  At  the  time  of  reproduction  or 
degeneration  it  not  infrequently  swells  up,  and  forms  a  viscous  hilum 
or  matrix,  inside  which  are  formed  the  new  sheaths  of  the  younger 
generation.  It  may  be  rigid,  and  so  maintain  the  normal  shape  of 
the  species,  or,  on  the  other  hand,  flexible,  and  so  adapted  to  rapid 
movement  of  the  individual. 

Here,  then,  we  have  the  major  parts  in  the  constitution  of  a 
bacillus — its  body,  mycoprotein ;  its  capsule,  cellulose.     But,  further 


f 


FIG.  3.— Diagrams  of  Normal  and  Polymorphic  Forms  of  Tubercle  Bacilli. 

than  this,  there  are  a  number  of  additional  distinctive  characteristics 
as  regards  the  contents  inside  the  capsule  which  call  for  mention. 
Sulphur  occurs  in  the  Beggiatoa  which  thrive  in  sulphur  springs. 
Starch  is  commoner  still.  Iron  as  oxide  or  other  combination  is 
found  in  several  species.  Many  contain  pigments,  though  these 
are  generally  the  "innocent"  bacteria,  in  contradistinction  to 
the  disease-producing.  A  pigment  has  been  found  which  is 
designated  lacterio-purpurin.  According  to  Zopf,  the  colouring 
agents  of  bacteria  are  the  same  as,  or  closely  allied  to,  the 
colouring  matters  occurring  widely  in  nature.  Migula  holds  that 
most  of  the  bacterial  pigments  are  non-nitrogenous  bodies.  There 
are  a  very  large  number  of  chromogenic  bacteria,  some  of  which 
produce  exceedingly  brilliant  colours.  Among  some  of  the  commoner 
forms  possessing  this  character  are  Bacillus  et  micrococcus  molaccus, 
B.  et  M.  aurantiacus  (orange) ;  B.  et  M.  luteus ;  M.  roseus  (pink) ; 
many  of  the  Sarcince ;  B.  aureus ;  B.  fluorescens  liqucfaciens  et 


BACTERIAL  POWERS  OF  MOTION  11 

non-liquefaciens  (green);  B.  pyocyaneus  (green);  B.  prodigiosus 
(blood-red). 

Motility. — When  a  drop  of  water  containing  bacteria  is  placed 
upon  a  slide,  a  clean  cover-glass*  superimposed,  and  the  specimen 
examined  under  an  oil  immersion  lens,  various  rapid  movements  will 
generally  be  observed  in  the  micro-organisms.  These  are  of  four 
chief  kinds :  (1)  A  dancing,  stationary  motion  known  as  Brownian 
movement.  This  is  molecular,  and  depends  in  some  degree  upon  heat 
and  the  medium  of  the  moving  particles.  It  is  non-progressive,  and 
is  well  seen  in  gamboge  particles.  (2)  An  undulatory,  serpentine 
movement,  with  apparently  little  advance  being  made.  (3)  A 
rotatory  movement,  which  in  some  water  bacilli  is  very  marked,  and 
consists  of  spinning  round,  sometimes  with  considerable  velocity,  and 
maintained  for  some  seconds  or  even  minutes.  (4)  A  progressive, 
darting  movement,  by  which  the  bacillus  passes  over  some  con- 
siderable distance. 

The  conditions  affecting  the  motility  of  bacteria  are  but  partly 
understood.  Heating  the  slide  or  medium  accelerates  all  movement. 
A  fresh  supply  of  oxygen,  or  indeed  the  addition  of  some  nutrient 
substance,  like  broth,  will  have  the  same  effect.  There  are  also  the 
somewhat  mysterious  powers  by  which  cells  possess  inherent 
attraction  or  repulsion  for  other  cells,  known  as  positive  and  negative 
ckemiotaxis.  These  powers  have  been  observed  in  bacteria  by 
Pfeiffer  and  Ali-Cohen. 

The  essential  condition  in  the  motile  bacilli  is  the  presence  of 
flagella*  These  cilia,  or  hairy  processes,  project  from  the  sides  or 
from  the  ends  of  the  rod,  and  are  freely  motile  and  elastic.  Some- 
times only  one  or  two  terminal  flagella  are  present ;  in  other  cases, 
like  the  bacillus  of  typhoid  fever,  five  to  twenty  may  occur  all  round 
the  body  of  the  bacillus,  varying  in  length  and  size,  sometimes 
being  of  greater  length  even  than  the  bacillus  itself.  It  is  not  yet 
established  as  to  whether  these  cilia  are  prolongations  of  capsule 
only,  or  whether  they  contain  something  of  the  body  protoplasm. 
Migula  holds  the  former  view,  and  states  that  the  position  of 
flagella  is  constant  enough  for  diagnostic  purposes.  They  are  but 
rarely  recognisable  except  by  means  of  special  staining  methods. 
Micfococcus  agilis  (Ali-Cohen)  is  one  of  the  rare  cases  of  a  coccus 
which  has  flagella  and  powers  of  active  motion. 

Modes  of  Reproduction. — Budding,  division,  and  spore  formation 
are  the  three  chief  ways  in  which  Schizomycetes  and  Saccharomycetes 
(yeasts)  reproduce  their  kind.  Budding  occurs  in  many  kinds  of 
yeast-cells,  and  generally  takes  place  when  the  nutriment  and 

*  A.flagellum  is  a  hair-like  process  arising  from  the  poles  or  sides  of  the  bacillus. 
It  must  not  be  confused  with  a,  filament,  which  is  a  thread-like  growth  of  the  bacillus 
itself. 


12  THE  BIOLOGY  OF  BACTERIA 

environment  are  favourable.  The  capsule  of  a  large,  or  "mother" 
cell,  shows  a  slight  protrusion  outwards,  which  is  gradually -enlarged 
into  a  "  daughter  "  yeast,  and  later  on  becomes  constricted  at  the  neck. 
Eventually  it  separates  as  an  individual.  The  protoplasm  of  the 
spores  of  yeasts  differs,  as  Hansen  has  pointed  out,  according  to  the 
conditions  of  culture. 

Division,  or  fission,  is  the  commonest  method  of  reproduction. 
It  occurs  transversely.  A  small  indentation  occurs  in  the  capsule, 
which  appears  to  make  its  way  slowly  through  the  whole  body  of  the 
bacillus  or  micrococcus  until  the  two  parts  are  separate,  and  each 
contained  in  its  own  capsule.  It  has  been  pointed  out  already  that 
in  the  incomplete  division  of  micrococci  we  observe  a  stage  precisely 
similar  to  a  diplococcus.  So  also  in  the  division  of  bacilli  an  appear- 
ance occurs  described  as  a  diplobacillus. 

Simple  fission  requires  but  a  short  period  of  time  to  be  complete. 
Hence  multiplication  is  very  rapid,  for  within  half  an  hour  a  new 
adult  individual  can  be  produced.  It  has  been  estimated  that  at  this 
rate  one  bacillus  will  in  twenty-four  hours  produce  millions  of  similar 
individuals;  or,  expressed  otherwise,  Cohn  calculated  that  in  three 
days,  under  favourable  circumstances,  the  rate  of  increase  would  be 
such  as  to  form  a  mass  of  living  organisms  weighing  many  tons,  and 
numbering  billions  of  individuals.  Favourable  conditions  do  not 
occur,  fortunately,  to  allow  of  such  increase,  which,  it  is  evident,  can 
only  be  roughly  estimated.  But  the  above  facts  illustrate  the 
enormous  fertility  of  micro-organic  life.  When  we  remember  that  in 
some  species  it  requires  10,000  or  15,000  fully-grown  bacilli  placed 
end  to  end  to  stretch  the  length  of  an  inch,  we  see  also  how  exceed- 
ingly minute  are  the  individuals  composing  these  unseen  hosts. 

Spore  formation  may  result  in  the  production  of  germinating  cells 
inside  the  capsule  of  the  bacillus,  endospores,  or  as  modified  individuals, 
arthrospores.  The  body  of  a  bacillus,  in  which  sporulation  is  about 
to  occur,  loses  its  homogeneous  character  and  becomes  granular,  owing 
to  the  appearance  of  globules  in  the  protoplasm.  In  the  course  of 
three  or  four  hours  the  globule  enlarges  to  fill  the  diameter  of  the 
rod,  and  assumes  a  more  concentrated  condition  than  the  parent  cell. 
At  its  maturity,  and  before  its  rupture  of  the  bacillary  capsule,  a 
spore  is  observed  to  be  bright  and  shining,  oval  and  regular  in  shape, 
with  concentrated  contents,  and  frequently  causing  a  local  expansion 
of  the  bacillus.  In  a  number  of  rods  lying  endwise,  these  local 
swellings  produce  a  beaded  or  varicose  appearance,  even  simulating 
a  streptococcus.  In  the  meantime  the  rod  itself  has  become  slightly 
broader  and  pale.  Eventually  it  breaks  down  by  segmentation  or 
by  swelling  up  into  a  gelatinous  mass.  The  spore  now  escapes  and 
commences  its  individual  existence.  Under  favourable  circumstances 
it  will  germinate.  The  tough  capsule  gives  way  at  one  point, 


MODES  OF  REPRODUCTION 


13 


O 


X 


generally  at  one  of  the  poles,  and  the  spore  sprouts  like  a  seed. 
In  the  space  of  about  one  hour's  time  the  oval  refractile  cell  has 
become  a  new  bacillus.  One  spore  produces  by  germination  one 
bacillus.  Spores  never  multiply  by  fission,  nor  reproduce  themselves. 

Hueppe  has  stated  that  there  are  certain  organisms  (like 
LeuconostoCy  and  some  streptococci)  which  reproduce  by  the  method 
of  arthrospores.  Defined  shortly,  this  is  simply  an  enlargement  of 
one  or  more  cell  elements  in  the  chain  which  thus  takes  on  the 
function  of  maternity.  On  either  side  of  the  large  coccus  may  be 
seen  the  smaller  ones, 
which  it  is  supposed  have 
contributed  of  their  proto- 
plasm to  form  a  mother 
cell.  An  arthrospore  is 
said  to  be  larger,  more  re- 
fractile, and  more  resistant 
than  an  ordinary  endospore. 
Many  bacteriologists  of  re- 
pute have  declined  hitherto 
definitely  to  accept  arthro- 
spore formation  as  a  proved 
fact. 

Spore  formation  in  bac- 
teria is  not  to  be  considered 
as  a  method  of  multipli- 
cation. The  general  rule 


is    undoubtedly   that   one 
bacillus  produces  one  spore, 

and    One    Spore   germinates     Flo>  ^-DIAGRAMS  OF  VARIOUS  FORMS  OF  SPORE  FORMATION 
illtO  0116    bacillus.       It    is  a  AKD  FLAGELLA. 

reproduction,  not  a  mul- 
tiplication. Indeed,  the 
whole  process  is  of  the  nature  of  a  resting  stage,  and  is  due  (a)  to  the 
arrival  of  the  adult  bacillus  at  its  biological  zenith,  or  (6)  to  the  con- 
ditions in  which  it  finds  itself  being  unfavourable  to  further  vegeta- 
tive growth,  and  so  it  endeavours  to  perpetuate  its  species.  Most 
authorities  are  probably  of  the  latter  opinion,  though  there  is  not  a 
little  evidence  for  the  former.  Exactly  what  conditions  are  favour- 
able to  sporulation  is  not  known.  Nutriment  has  probably  an 
intimate  effect  upon  it.  The  temperature  must  not  be  below  16°  C., 
nor  much  above  40°  C.  Oxygen,  as  we  have  seen,  is  favourable,  if 
not  necessary,  to  many  species,  which  will  in  cultivation  in  broth 
rise  to  the  surface  and  lodge  in  the  pellicle  to  form  their  seeds. 
Moisture,  too,  is  considered  a  necessity. 

Koch  found  that  spore  formation  in  B.  anthracis  occurred  in  six 


A.  Stages  in  formation  of  spore  and  its  after  development. 
B.  Spirillum  with  terminal  flagella. 


14  THE  BIOLOGY  OF  BACTERIA 

hours.  The  spores  may  be  situated  in  the  middle  of  the  bacillus 
(as  in  B.  anthracis,  B.  acidi  lutyrici,  etc.),  towards  one  end  (Bacillus 
of  Malignant  (Edema),  or  actually  terminal  (B.  tetani).  Those  spores 
produced  inside  the  capsule  of  the  bacillus  are  termed  endospores. 
Hueppe  has  described  the  spores  of  certain  streptococci  as  arthrospores. 
The  spores  of  yeast  are  termed  ascospores.  The  spores  of  all 
bacillary  species  possess,  however,  certain  characters  in  common. 
They  are  as  follow.  The  spore  is  generally  oval,  though  more 
spherical  in  the  Hyphomycetes :  it  is  bright  and  glistening  in  aspect ; 
it  is  often  greater  in  diameter  than  the  bacillus  giving  rise  to  it ; 
its  capsule  is  thicker  and  stronger  than  the  capsule  of  the  parent 
bacillus;  and  it  is  generally  held  that  the  contained  protoplasm  is 
more  concentrated,  so  to  speak,  than  that  of  the  bacillus.  These  two 
last  characters  are  of  chief  importance  to  us,  for  it  is  owing  to  them 
that  spores  possess  such  marked  power  of  resistance.  Cohn  has 
suggested  that  the  capsule  of  a  spore  is  in  reality  a  double  envelope, 
an  inner  one  of  fatty  and  an  outer  one  of  gelatinous  nature,  and  it  is 
owing  to  this  that  its  resistance  to  heat  and  dessication  is  due.  The 
protoplasm  of  the  spore  contains,  of  course,  the  essential  constituents 
of  the  mother  cell.  It  is  the  method  by  which  "the  continuity  of 
germ  plasm  "  is  secured  in  these  lowly  forms  of  life.  Under  favourable 
circumstances  this  spore-protoplasm  will  germinate  into  a  new  bacillus. 
It  should  be  understood  that  whilst  holding  the  view  that 
spores  are  a  resting  stage  during  adverse  conditions,*  we  fully 
recognise  that  certain  favouring  external  conditions  are  essential 

*  Yeast  can  be  effectually  starved  by  cultivating  on  a  small  block  of  plaster- 
of-Paris  kept  moist  under  a  bell  jar ;  under  these  circumstances  the  yeast  is 
supplied  with  nothing  but  water.  In  a  few  days  the  protoplasm  of  yeast  cells  thus 
circumstanced  becomes  filled  with  vacuoles  and  fat  cells.  The  protoplasm  has 
been  undergoing  destructive  metabolism,  and,  there  being  nothing  to  supply  new 
material,  has  diminished  in  quantity  and  at  the  same  time  been  partly  converted 
into  fat.  Both  in  plants  and  animals  fatty  degeneration  is  a  more  or  less  constant 
phenomenon  of  starvation,  and  to  this  bacteria  are  no  exception.  After  a  time  the 
protoplasm  collects  towards  the  centre  of  the  cell,  and  divides  simultaneously  into 
four  masses  arranged  like  a  pyramid  of  four  billiard  balls,  three  at  the  base  and 
one  above.  These  are  the  ascospores,  and  sooner  or  later  they  are  liberated  by 
the  rupture  of  the  mother-cell  wall.  Certain  of  the  Streptothrix  family  also 
"  sporulate "  when  they  find  themselves,  like  yeast  upon  gypsum,  surrounded 
by  an  unfavourable  environment.  Again,  in  old  cultures,  it  will  be  found  that 
when  the  food  supply  has  been  exhausted  the  bacteria  have  either  sporulated  or 
have  died.  For  these  reasons  sporulation  may  be  looked  upon  not  as  a  method 
of  multiplication  but  one  of  reproduction,  of  carrying  on  the  species  under  adverse 
conditions.  With  regard  to  the  rapid  formation  of  spores  under  apparently 
favourable  circumstances  (B.  filamentosus^  B.  anthracis,  etc. ),  it  must  be  borne  in 
mind  that  the  medium  may  not  be  by  any  means  so  favourable  as  appears  to  be  the 
case  (Flugge).  It  is  clear  that  the  food  supply  immediately  around  many  of  the 
bacteria  in  a  culture  must  soon  be  exhausted.  Besides,  there  is  the  toxic  influence 
early  at  work,  often  as  an  inimical  agency  acting  unfavourably  towards  the  bacillus 
producing  it.  So  that  the  appearance  of  spores  in  such  a  culture  may  still  be  due 
to  conditions  which  are  actually  unfavourable. 


INFLUENCE  OF  EXTERNAL  CONDITIONS  15 

to  spore  formation.  Of  these,  there  are  at  least  three  of  which 
bacteriologists  have  knowledge,  namely,  moisture,  oxygen,  and  a 
certain  temperature.  Fluid  media  forms  an  excellent  nidus  for 
sporulation  so  long  as  some  oxygen  can  gain  access  to  the  sporulating 
germs.  But  many  organisms  will  not  sporulate  if  lying  deep  in 
such  a  medium.  In  moulds  and  yeasts  oxygen  is  essential,  and  for 
some  spore-bearing  bacilli  a  supply  of  oxygen  is  a  sine  qud  non  (the 
exceptions  are  strict  anaerobes  like  B.  tetani,  B.  butyricus,  etc.)  of 
sporulation.  Prazmowski  has  pointed  out  that  it  is  characteristic  of 
these  forms  that  they  are  non-motile  during  sporulation.  B.  tetani, 
B.  lutyricus,  and  other  strict  anaerobes  continue  to  remain  motile 
during  sporing.  Temperature  exerts  a  marked  influence  on  the 
process.*  In  the  case  of  B.  subtilis,  an  organism  frequently  present  in 
milk,  spore  formation  did  not  occur  below  6°  C. ;  at  18°  C.  it  required 
two  days ;  at  22°  C.  one  day ;  and  at  30°  C.  only  twelve  hours,  f  • 

When  free  in  the  field  of  the  microscope,  spores  must  be  dis- 
tinguished from  fat  cells,  micrococci,  starch  cells,  some  kinds  of  ova, 
yeast  cells,  and  other  like  objects.  Spores  are  detected  frequently 
by  their  resistance  to  ordinary  stains  and  the  necessity  of  colouring 
them  by  special  staining  methods.  When,  however,  a  spore  has 
taken  on  the  desired  colour,  it  retains  it  with  tenacity.  In  addition 
to  their  shape,  size,  thickened  capsule,  and  staining  characteristics, 
spores  also  resist  desiccation  and  heat  in  a  much  higher  degree  than 
bacilli  not  bearing  spores.  It  has  been  suggested  that  bacteria 
should  be  classified  according  to  their  method  of  spore  formation. 

The  Influence  of  External  Conditions  on  the 
Growth  of  Bacteria 

In  the  earliest  days  of  the  study  of  micro-organisms  it  was 
observed  that  they  mostly  congregate  where  there  is  suitable  food 
for  their  nourishment.  The  reason  why  fluids  such  as  milk,  and 
dead  animal  matter  such  as  a  carcase,  and  living  tissues  such  as  a 
man's  body,  contain  many  microbes,  is  because  each  of  these  three 
media  is  favourable  to  their  growth.  Milk  affords  almost  an  ideal 
food  and  environment  for  microbes.  Its  temperature  and  con- 
stitution frequently  meet  their  requirements.  Dead  animal  matter, 
too,  yields  a  rich  diet  for  certain  species  (saprophytes).  In  the 
living  tissues  bacteria  obtain  not  only  nutriment,  but  a  favourable 

*  Koch  has  shown  in  the  case  of  B.  anthracis  that  at  least  16°  C.  is  necessary 
for  spore  formation,  and  at  this  temperature  limited  formation  of  spores  did  not 
occur  until  after  seven  days.  At  21°  C.  spores  had  formed  after  seventy-two  hours, 
at  25°  C.  after  thirty- five  to  forty  hours,  and  between  30°  C.  and  40°  C.  in  about 
twenty-four  hours ;  •  the  best  and  strongest  cultivations  were  obtained  from  20° 
to  25°  C. 

f  Fliigge. — Micro-organisms.     Translation  by  W.  Watson  Cheyne,  1890,  p.  539. 


16  THE  BIOLOGY  OF  BACTERIA 

temperature  and  moisture.  Outside  the  human  body  it  has  been  the 
endeavour  of  bacteriologists  to  provide  media  as  similar  to  the  above 
as  possible,  and  containing  many  of  the  same  elements  of  food,  in 
order  that  the  life-history  may  be  carried  on  outside  the  body  and 
under  observation.  By  means  of  cover-glass  preparations  for  the 
microscope  we  are  able  to  study  the  form,  size,  motility,  flagella, 
spore  formation,  and  peculiarities  of  staining,  all  of  which  characters 
aid  us  in  determining  to  what  species  the  organism  under  examination 
belongs.  By  means  of  artificial  nutrient  media  we  may  further 
learn  the  characters  of  the  organism  in  "  pure  culture,"  *  its  favour- 
able temperature,  its  power  or  otherwise  of  liquefaction,  of  curdling 
of  milk,  or  of  gas  or  acid  production ;  its  behaviour  towards  oxygen ; 
its  power  of  producing  indol,  pigment,  and  other  bodies ;  as  well  as 
its  thermal  death-point  and  resistance  to  light  and  disinfectants.  It 
is  well  known  that  under  artificial  cultivation  an  organism  may  be 
greatly  modified  in  its  morphology  and  physiology,  and  yet  its 
conformity  to  type  remains  much  more  marked  than  any  divergence 
which  may  occur. 

Nutritive  Medium.f  The  basis  of  many  of  these  artificial  media  is  broth. 
This  is  made  from  good  lean  beef,  free  from  fat  and  gristle,  which  is  finely  minced 
up  and  extracted  in  sterilised  water  (one  pound  of  lean  beef  to  every  1000  c.c.  of 
water).  It  is  then  filtered  and  sterilised.  To  provide  peptone  beef-broth,  ten 
grammes  of  peptone  and  five  grammes  of  common  salt  are  added  to  every  litre  of 
acid  beef-broth.  It  is  rendered  slightly  alkaline  by  the  addition  of  sodium  car- 
bonate or  sodium  hydrate,  and  is  filtered  and  sterilised.  In  glycerine-broth  6  to  8 
per  cent,  of  glycerine  has  been  added  after  filtration,  in  glucose-broth  1  or  2  per  cent, 
of  grape-sugar.  This  latter  is  used  for  anaerobic  organisms.  The  use  of  broth  as 
a  culture  medium  is  of  great  value.  It  is  undoubtedly  the  best  fluid  medium,  and 
in  it  may  not  only  be  kept  pure  cultures  of  bacteria  which  it  is  desired  to  retain  for 
a  length  of  time,  but  in  it  also  emulsions  and  mixtures  may  be  placed  preparatory  to 
further  examination.  Gelatine  consists  of  broth  solidified  by  the  addition  of  100 
grams  of  best  French  gelatine  to  the  litre.  Its  advantage  is  twofold :  it  is  trans- 
parent, and  it  allows  manifestation  of  the  power  of  liquefaction.  When  we  speak 
of  a  liquefying  organism  we  mean  a  germ  having  the  power  of  producing  a  pepton- 
ising  ferment  which  can  at  the  temperature  of  the  room  break  down  solid  gelatine 
into  a  liquid.  Grape-sugar  gelatine  is  made  like  grape-sugar  broth.  Agar  was 
introduced  as  a  medium  which  would  not  like  gelatine  melt  at  25°  C. ,  but  remain 
solid  at  blood-heat  (37'5°  C. ;  98 '5°  F.).  It  is  a  seaweed  generally  obtained  in  dried 
strips  from  the  Japanese  market.  Ten  to  fifteen  grammes  are  added  to  every  litre 
of  peptone-broth.  Glycerine  and  grape-sugar  may  be  added  as  elsewhere.  Blood 
agar  is  ordinary  agar  with  fresh  sterile  blood  smeared  over  its  surface.  Blood  serum 
is  drawn  from  a  jar  of  coagulated  horse-blood,  in  which  the  serum  has  risen  to  the 
top.  This  is  collected  in  sterilised  tubes  and  coagulated  in  a  special  apparatus  (the 
serum  inspissator).  Potato  is  prepared  by  scraping  ordinary  potatoes,  washing  in 
corrosive  sublimate,  and  sterilising.  It  may  then  be  cut  into  various  shapes  con- 
venient for  cultivation.  Upon  any  of  these  forms  of  solid  media  the  characteristic 

*  A  "pure  culture"  is  a  growth,  in  an  artificial  medium  outside  the  body,  of  one 
species  of  micro-organism  only. 

f  The  facts  here  given  are  obviously  only  general  indications.  The  accurate 
preparation  of  medium  is  of  vital  importance  in  Bacteriology,  and  for  its  accomplish- 
ment text-books  should  be  consulted  (Eyre's  Bacteriological  Technique,  1 25-1 74). 


TEMPERATURE 


17 


growth   of  the   organism  can   be  observed.      Of  the  nutrient  elements  required, 
nitrogen    is    obtained    from    albumens    and    proteids,    carbon    from    milk-sugar, 
cane-sugar, «or  the  splitting  up  of  proteids;    salts  (particularly  phosphates  and 
salts   of  potassium)  are  readily  obtain- 
able   from    those    incorporated    in    the  f~ 

media ;  and  the  water  which  is  required     ^^  V_ 

is  obtainable  from  the  moisture  of  the 
media. 


FIG.  5. — INOCULATING  NEEDLES. 
Platinum  wire  fused  into  glass  handles.. 


There  are  two  common  forms 

of  test-tube  culture,  viz.,  on  the 

surface  and  in  the  depth  of  the 

medium.      In     the    former    the 

medium  is  sloped,  and  the  inocu- 
lating needle  is  drawn  along  its 

surface ;  in  the  latter  the  needle 

is    thrust   vertically   downwards 

into     the    depth     of     the    solid 

medium.     Plate  cultures  and  anaerobic  cultures  will  be  described  at 

a  later  stage. 

Temperature. — -When   the   medium,    has   been  inoculated    the 

culture  is  placed  at  a  temperature  which  will  be  favourable.  For 
every  species  of  bacteria  there  is  a  favour- 
able temperature,  termed  the  optimum 
temperature.  This  is  usually  the  tempera- 
ture of  the  natural  habitat  of  the  organism. 
Two  standards  of  temperature  are  in  use  in 
bacteriological  laboratories.  The  one,  room 
temperature,  varies  from  18°-22°  C. ;  the 
other  is  Hood-heat,  and  varies  from  35°-38°  C. 
(Plates  1  and  2).  It  is  true  some  species  will 
grow  below  18°  C.,  and  others  above  38°  C. 
The  pathogenic  (disease-producing)  bacteria 
thrive  best  as  a  rule  at  37°  C.,  and  the  non- 
pathogenic  at  the  ordinary  temperature  of 
the  room.  The  different  degrees  of  tempera- 
ture are  obtained  by  means  of  incubators. 
For  the  low  temperatures  gelatine  is  chosen 
as  a  medium,  for  the  higher  temperatures 
agar.  Most  bacteria  grow  well  at  room 
temperature  (about  60°  F.),  but  they  will 
grow  more  luxuriantly  and  speedily  at 
blood-heat. 
Whilst  these  are  the  ordinary  limits  of  temperature  affecting 

bacteria,  they  do  not  by  any  means  include  the  extremes  of  heat  and 

cold  which  micro-organisms   can  withstand.      The   average  thermal 

death-point  is  about  55°  C,  but  certain  species,  termed  thermophilic, 

E 


FIG.  6.— Media  for 'Surface 
and  Depth  Culture. 


18  THE  BIOLOGY  OF  BACTERIA 

isolated  from  the  intestine,  horse  manure,  etc.,  grow  at  60°-70°  C. 
On  the  other  hand,  investigations  have  shown  that  bacteria  can 
withstand  exceedingly  low  temperatures.  Koch  showed  that  the 
cholera  vibrio  was  not  killed  by  a  temperature  of  —32°  C.  In  1900, 
Swithinbank  exposed  cultures  of  the  tubercle  bacillus  to  the 
temperature  of  liquid  air  (—193°  C.)  for  continuous  periods  varying 
from  six  hours  to  forty-two  days,  without  their  vitality  being  affected ; 
and  in  the  same  year  MacFadyen  and  Eowland  found  that  Proteus 
vulgaris,  B.  coli,  and  several  other  species  were  not  killed  after  an 
exposure  of  ten  hours  to  a  temperature  of  liquid  hydrogen  (  —  252°  C). 
It  will  thus  be  seen  that  bacteria  can  withstand  great  alternations  of 
temperature.  From  a  public  health  point  of  view,  it  is  important 
to  remember  that  organisms  can  exist  in  freezing  mixtures  and  ice, 
retaining  their  vitality  and  virulence.  For  example,  B.  coli  and  the 
typhoid  bacillus  can  exist  from  the  low  temperatures  above  mentioned 
to  80°  C.,  although  the  usual  thermal  death-point  for  these  species 
is  between  50°-60°  C.* 

Moisture  has  been  shown  to  have  a  favourable  effect  upon  the 
growth  of  microbes.  Drying  will  of  itself  kill  many  species  (e.g.  the 
spirillum  of  cholera),  and  other  things  being  equal,  the  more  moist  a 
medium  is,  the  better  will  be  the  growth  upon  it.  Thus  it  is  that  the 
growth  in  broth  is  always  more  luxuriant  than  that  on  solid  media. 
Yet  the  growth  of  Bacillus  subtilis  and  some  other  species  are  an 
exception  to  this  rule,  for  they  prefer  a  dry  medium.  Desiccation  as 
a  rule  diminishes  virulence  and  lessens  growth.  But  some  species 
can  withstand  long-continued  drying  without  injury. 

Light  acts  as  an  inhibitory,  or  even  germicidal,  agent.  This 
fact  was  first  established  by  Downes  and  Blunt  in  a  memoir  to  the 
Eoyal  Society  in  1877.  They  found  by  exposing  cultures  to  different 
degrees  of  sunlight  that  the  growth  of  the  culture  was  partially  or 
entirely  prevented,  being  most  damaged  by  the  direct  rays  of  the 
sun,  although  diffuse  daylight  acted  prejudicially.  Further,  these 
same  investigators  proved  that  the  rays  of  the  spectrum  which  acted 
most  inimically  upon  bacteria  were  the  blue  and  violet  rays,  next  to 
the  blue  being  the  red  and  orange-red  rays.  The  action  of  light, 
they  explain,  is  due  to  the  gradual  oxidation  which  is  induced  by  the 
sun's  rays  in  the  presence  of  oxygen.  Duclaux,  who  worked  at  this 
question  at  a  later  date,  concluded  that  the  degree  of  resistance  to 
the  bactericidal  influence  of  light,  which  some  bacteria  possess, 
might  be  due  to  difference  in  species,  difference  in  culture  media,  and 
difference  in  the  degrees  of  intensity  of  light.  Tyndall  tested  the 
growth  of  organisms  in  flasks  exposed  to  air  and  light  on  the  Alps, 

*  For  the  latest  researches  on  this  point,  see  Proc.  Roy.  Soc.,  1900  and  1901  ;  and 
the  Thirty-fourth  Annual  Report  of  the  State  Board  of  Health,  Massachusetts,  1903, 
pp.  269-281.  Dewar  commenced  experiments  of  this  character  in  1892. 


A  FORM  OF  PASTEITR'S  LARGE  INCUBATOR  FOR  CULTIVATION  AT  ROOM  TEMPERATURE. 


[To  face  page  18. 


EFFECT  OF  LIGHT  19 

and  found  that  sunlight  inhibited  the  growth  temporarily.  A  large 
number  of  experimenters  on  the  Continent  and  in  England  have 
worked  at  this  fascinating  subject  since  1877,  and  though  many  of 
their  results  appear  contradictory,  we  may  be  satisfied  in  adopting 
the  following  conclusions  respecting  the  matter : — 

(1)  Sunlight  has  a  deleterious  effect  upon  bacteria,  and  to  a  less 
extent  on  their  spores. 

(2)  This  inimical  effect  can  be  produced  by  light  irrespectively  of 
rise  in  temperature. 

(3)  The   ultra-violet   rays  are   the    most   bactericidal,   and   the 
infra-red  the  least  so,  which  indicates  that  the  phenomenon  is  due 
to  chemical  action. 

(4)  The  presence  of  oxygen  and  moisture  greatly  increase  this 
action,  the  process  being  largely  an  oxidation. 

(5)  Sunlight  also  acts  prejudicially  upon  the  culture  medium, 
and  thereby  exerts  an  injurious  action  on  the  culture. 

(6)  The    time     occupied    in    the    bactericidal    action    depends 
upon  the  intensity  of  the  light  and  tne  inherent   vitality  of  the 
organism. 

(7)  With  regard  to  the  action  of  light  upon  pathogenic  organisms, 
some  results  have  recently  been  obtained   with   Bacillus   typJiosus. 
Janowski  maintains  that  direct  sunlight  exerts  a  distinctly  depressing 
effect  on  typhoid  bacilli.     At  present  more  cannot  be  said  than  that 
sunlight  and  fresh  air  are  two  of  the  most  powerful  agents  we  possess 
with  which  to  combat  pathogenic  germs. 

A  very  simple  method  of  demonstrating  the  influence  of  light 
is  to  grow  a  pure  culture  in  a  favourable  medium,  either  in  a  test- 
tube  or  upon  a  glass  plate,  and  then  cover  the  whole  with  black 
paper  or  cloth.  A  little  window  may  then  be  cut  in  the  protec- 
tive covering,  and  the  whole  exposed  to  the  light.  Where  it 
reaches  in  direct  rays,  it  will  be  found  that  little  or  no  growth  has 
occurred;  where,  on  the  other  hand,  the  culture  has  been  in  the 
dark,  abundant  growth  occurs.  In  diffuse  light  the  growth  is 
merely  somewhat  inhibited. 

A  number  of  experiments  in  this  direction  were  made  at  Lawrence, 
Massachusetts,*  with  cultures  of  typhoid  and  B.  coli. 

In  two  experiments,  each  with  typhoid  bacillus  and  B.  coli,  water 
dilutions  were  made  from  fresh  cultures  of  the  germs,  1  c.c.  of  this 
water  being  placed  in  Petri  dishes  in  the  sun  for  definite  periods. 
After  exposure,  the  water  in  the  plates  was  mixed  with  agar,  and  all 
plates  were  incubated  twenty-four  hours  at  38°,  after  which  the 
number  of  colonies  was  counted.  In  one  experiment  the  water 
dilution  of  typhoid  was  mixed  with  melted  agar,  and  plates  made  as 

*  Thirty -fourth    Ann.    Rep.    State    Ed.    of  Ilealth    of    Massachusetts,    1903, 
p.  275. 


20 


THE  BIOLOGY  OF  BACTERIA 


usual.  After  the  agar  had  set,  these  plates  were  then  exposed  to  the 
sunlight.  In  one  experiment  with  B.  coli,  the  water  culture  was  not 
exposed  to  the  sunlight  in  plates,  but  the  exposure  was  made  in  a 
clear,  white  glass  bottle  of  the  Blake  pattern,  holding  100  c.c., 
samples  being  taken  from  this  at  the  proper  intervals,  and  plated 
as  usual. 

In  all  cases  control  cultures  were  made  under  exactly  the  same 
conditions  as  were  the  cultures  exposed,  these,  however,  being  pro- 
tected from  the  sunlight  by  a  heavy,  opaque  cloth,  or  some  similar 
material.  The  temperature  of  these  cultures  was,  of  course,  consider- 
ably lower  than  was  the  temperature  in  the  sun.  The  numbers  of 
bacteria  in  the  controls  showed  the  usual  variation  to  be  expected 
under  the  circumstances,  usually  a  slight  reduction  in  numbers  being 
noted  during  two  or  three  hours'  standing,  although  in  one  instance 
the  numbers  increased  quite  materially.  The  data  of  these  control 
cultures  are  not  shown  in  the  accompanying  tables. 

The  brightness  of  the  sun  also  varied  considerably,  and  attempts 
were  made  to  measure  the  amount  of  light  by  photographic  means, 
but  these  measurements  were  unsatisfactory,  and  the  data  are  not 
included  here. 

With  typhoid,  from  95  to  99  per  cent,  of  all  the  germs  were 
destroyed  by  ten  to  fifteen  minutes'  exposure  to  direct  sunlight.  A 
few  germs  may  resist  the  sunlight  for  a  somewhat  longer  time ; 
usually,  however,  all  the  germs  were  destroyed  by  three  or  more 
hours'  exposure  to  bright  sunlight.  The  results  of  the  experiments 
with  typhoid  are  shown  in  the  following  tables : — 


TABLE  showing  Elimination  of  Typhoid  Germs  in   Water  on 
Exposure  to  Sunlight. 


Experiment  24. 

Experiment  25. 

Bacteria. 

Average. 

Bacteria. 

Average. 

Start       . 

698     734 

716 

592     532 

562 

15  minutes 

66         4 

35 

13         5 

9 

30  minutes 

17         1 

9 

4         4 

4 

45  minutes 

0         1 

1 

3         0 

2 

1  hour    . 

6         2 

4 

21         5 

13 

H  hours 

2         0 

1 

5         3 

4 

2  hours  . 

0         0 

0 

4         3 

4 

4  hours  . 

3         1 

2 

0         0 

0 

6  hours  . 

0         0 

0 

0         0 

0 

EFFECT  OF  LIGHT 


21 


TABLE  showing  Elimination  of  Typhoid  Germs  in  A  gar 
Platen  on  Exposure  to  Sunlight. 


Exposure. 

Temperature. 

Experiment  26. 

Bacteria. 

Average. 

Start    .                    ^ 

608     642 

625 

10  minutes 

91°  F. 

2         3 

3 

20  minutes 

83 

7         2 

5 

30  minutes 

81 

0         0 

0 

40  minutes 

83 

0         0 

0 

50  minutes 

83                   00 

0 

1  hour  . 

78 

1         0 

1 

1  hour,  10  minutes 

74                   00 

0 

1  hour,  20  minutes 

71                   00 

0 

1  hour,  30  minutes 

69 

0         0 

0 

1  hour,  40  minutes 

68 

o       o 

0 

1  hour,  50  minutes 

71 

0         0 

0 

2  hours 

68 

0         0 

0 

With  B.  coli  the  results  have  been  somewhat  more  variable,  prob- 
ably due  to  more  changeable  conditions.  In  one  experiment,  some- 
thing over  80  per  cent,  of  the  germs  were  destroyed  by  fifteen 
minutes'  exposure,  all  being  destroyed  after  four  hours.  The  results 
of  this  experiment  were  undoubtedly  influenced  greatly  by  clouds 
in  the  sky,  so  that  at  times  the  sunlight  was  not  very  bright,  after 
about  two  and  one-half  hours  the  sun  being  entirely  overcast.  In 
one  experiment  about  96  per  cent,  of  the  germs  were  eliminated  at 
the  end  of  fifteen  minutes,  and  after  thirty  minutes  all  of  the  germs 
were  destroyed.  In  these  two  experiments  the  water  cultures  were 
exposed  in  plates,  the  results  being  shown  in  the  following  table : — 

TABLE  showing  Elimination  of  B.  coli  in   Water  Cultures  on 
Exposure  to  Sunlight. 


Experiment  78. 

Experiment  79. 

Exposure. 

Tem- 
perature. 

Bacteria 
per  c.c. 

Average. 

Tem- 
perature. 

Bacteria 
per  c.c. 

Average. 

Start  . 

78 

70,000    72,800 

71,400 

445,400  824,300 

634,850 

15  minutes 

78 

6,564   16,166 

12,365 

106 

19,100     31,800 

25,400 

30  minutes 

80 

4,473     2,663 

3,568 

107 

0              0 

0 

45  minutes 

80 

2,130             0 

1,065 

110 

0              0 

0 

1  hour 

80 

590             0 

295 

100 

0              0 

0 

1  .V  hours 

82 

2,130           93 

1,111 

108 

0              0 

0 

2  hours 

62 

10          70 

40 

105 

0              0 

0 

3  hours 

100 

0              0 

0 

4  hours 

61 

0            0 

0 

78 

0              0 

0 

22 


THE  BIOLOGY  OF  BACTERIA 


In  one  experiment  the  water  was  exposed  in  bottles.  In  this 
case  about  98  per  cent,  of  the  germs  were  destroyed  after  fifteen 
minutes,  the  cultures  varying  somewhat.  The  germs  persisted  in 
the  water  in  considerable  numbers  for  two  hours  and  in  small 
numbers  up  to  four  hours,  after  five  hours  the  sample  being 
completely  sterilised.  The  results  of  this  experiment  are  shown 
as  follows : — 

TABLE  shotting  Change  in  Numbers  of  B.  coli  in   Water  in 
Bulk  on  Exposure  to  Sunlight. 


Experiment  81 

Exposure. 

Temperature. 

Bacteria  per 
c.c. 

Average. 

Start      . 

94 

2,360,000     1,620,000 

1,990,000 

15  minutes 

94 

30,000          43,200 

36,600 

30  minutes 

92 

85,300          22,000 

53,650 

45  minutes 

92 

44,000          55,000 

49,500 

1  hour    . 

94 

53,700          45,300 

49,500 

1^  hours 

96 

35,800          34,100 

34,950 

2  hours  . 

109 

57,400          76,400 

66,900 

3  hours  . 

95 

450            1,172 

786 

4  hours  . 

102 

3                   5 

4 

5  hours  . 

76 

0                   0 

0 

It  has  been  found  that  the  electric  light  has  but  little  action  upon 
bacteria,  though  that  which  it  has  is  similar  to  sunlight.  Eecent 
experiments  with  the  Rontgen  rays  have  not  given  bactericidal 
results. 

In  1890  Koch  stated  that  tubercle  bacilli  were  killed  after  an 
exposure  to  direct  sunlight  of  from  a  few  minutes  to  several  hours. 
The  influence  of  diffuse  light  would  obviously  be  much  less.  Professor 
Marshall  Ward  has  experimented  with  the  resistant  spores  of 
Bacillus  anthracis  by  growing  these  on  agar  plates  and  exposing  to 
sunlight.  From  two  to  six  hours'  exposure  had  a  germicidal  effect.* 

It  should  be  remembered  that  several  species  of  sea-water  bacteria 
themselves  possess  the  property  of  phosphorescence.  Pfliiger  was  the 
first  to  point  out  that  it  was  such  organisms  which  provided  the 
phosphorescence  upon  decomposing  wood  or  decaying  fish.  To  what 
this  light  is  due,  whether  capsule,  or  protoplasm,  or  chemical  product, 
is  not  yet  known.  The  only  facts  at  present  established  are  to  the 
effect  that  certain  kinds  of  media  and  pabulum  favour  or  deter 
phosphorescence. 

*  See  Trans.  .Tenner  Inst.  (second  series),  1899,  p.  81. 


MEANS  OF  STERILISATION 


23 


Aerobiosis. — Pasteur  was  the  first  to  lay  emphasis  upon  the 
effect  which  free  air  had  upon  micro-organisms.  He  classified  them 
according  to  whether  they  grew  in  air,  aerobic,  or  whether  they 
flourished  most  without  it,  anaerobic.  Some  have  the  faculty  of 
growing  with  or  without  the  presence  of  oxygen,  and  are  designated 
SLS  facultative  aerobes  or  anaerobes.  As  regards  the  cultivation  of 
anaerobic  germs,  it  is  only  necessary  to  say  that  hydrogen,  nitrogen, 
or  carbonic  acid  gas  may  be  used  in  place  of  oxygen,  or  they  may 


FIG.  7.— Method  of  producing  Hydrogen  by  Kipp's  Apparatus  for  Cultivation  of 
Anaerobes  (see  p.  117. 

be  grown  in  a  medium  containing  some  substance  which  will  absorb 
the  oxygen  (see  p.  117). 

Means  of  Sterilisation. — As  this  term  occurs  frequently  even 
in  books  of  an  elementary  nature,  and  as  it  is  expressive  of  an  idea 
which  must  always  be  present  to  the  mind  of  the  bacteriologist,  it 
may  be  desirable  to  make  allusion  to  it  here. 

Chemical  substances,  perfect  filtration,  and  heat  are  the  three 
means  at  our  command  in  order  to  secure  germ-free  conditions  of 
apparatus  or  medium.  The  first  two,  though  theoretically  admissible, 
are  practically  seldom  used,  the  former  of  the  two  because  the 
addition  of  chemical  substances  annuls  or  modifies  the  operation, 
the  latter  of  the  two  on  account  of  the  great  practical  difficulties  in 
securing  efficiency.  Hence  in  the  investigations  involved  in 
bacteriological  research  heat  is  the  common  sterilising  agent.  A 
sustained  temperature  of  70°  C.  (158°  F.)  will  kill  all  bacilli;  even 
58°  C.  will  kill  most  kinds.  Boiling  at  100°  C.  (212°  F.)  for  five 
minutes  will  kill  anthrax  spores,  and  for  thirty  to  sixty  minutes 
will  kill  all  bacilli  and  their  spores.  This  difference  in  the  thermal 


24 


THE  BIOLOGY  OF  BACTERIA 


death-point  between  bacilli  and  their  spores  enables  the  operator  to 
obtain  what  are  called  "pure  cultures"  of  a  desired  bacillus  from 
its  spores  which  may  be  present.  For  example,  if  a  culture  contains 

spores  of  anthrax  and  is  contaminated 
with  micrococci,  heating  to  70°  C. 
(158°  F.)  will  kill  all  the  micrococci, 
but  will  not  affect  the  spores  of  an- 
thrax, which  can  then  grow  into  a 
pure  culture  of  anthrax  bacilli.  Frac- 
tional or  discontinuous  sterilisation 
depends  on  the  principle  of  heating 
to  the  sterilising  point  for  bacilli  (say 
70°  C.)  on  one  day,  which  will  kill  the 
bacilli,  but  leave  the  spores  uninjured. 
But  by  the  following  day  the  spores 
will  have  germinated  into  bacilli,  and 
a  second  heating  to  70°  C.  will  kill 
them  before  they  in  their  turn  have 
had  time  to  sporulate.  Thus  the 
whole  will  be  sterilised,  though  at  a 
temperature  below  boiling. 

Successful  sterilisation,  therefore, 
depends  upon  killing  both  bacteria 
and  their  spores,  and  nothing  short 
of  that  can  be  considered  as  sterilisa- 
tion. The  following  methods  are 
those  generally  used  in  the  laboratory. 
For  dry  heat  (which  is  never  so  in- 
jurious to  organisms  as  moist  heat*) : 
(a)  the  Bunsen  burner,  in  the  flame  of 
which  platinum  needles,  etc.,  are  steril- 
ised; (b)  hot-air  chamber,  in  which 
flasks  and  test-tubes  are  heated  to  a  temperature  of  150°-170°  C. 
for  an  hour  or  more.  For  moist  heat :  (c)  boiling,  for  knives  and 

*  It  will  be  observed  that  there  is  a  marked  difference  between  the  effects  of  dry 
heat  and  moist  heat.  Moist  heat  is  able  to  kill  organisms  much  more  readily  than 
dry,  owing  to  its  penetrating  effect  on  the  capsule  of  the  bacillus.  Dry  heat  at 
140°  C.  (284°  F.),  maintained  for  three  hours,  is  necessary  to  kill  the  resistant  spores 
of  Bacillus  anthracis  and  B.  subtilis,  but  moist  heat  at  forty  degrees  less  will  have  the 
same  effect.  It  is  from  data  such  as  these  that  in  laboratories  and  in  disinfecting 
apparatus  moist  heat  is  invariably  preferred  to  dry  heat  For  with  the  latter  such 
high  temperatures  would  be  required  that  the  articles  being  disinfected  would  be 
damaged.  Koch  states  the  following  figures  for  general  guidance :  Dry  heat  at  a 
temperature  of  120°  C.  (248°  F.)  will  destroy  spores  of  mould  fungi,  micrococci,  and 
bacilli  in  the  absence  of  their  spores  ;  for  the  spores  of  bacilli  140°  C.  (284°  F.), 
maintained  for  three  hours,  is  necessary;  moist  heat  at  100°  C.  (212°  F.)  for  fifteen 
minutes  will  kill  bacilli  and  their  spores. 


FIG.  8.— Koch's  Steam  Steriliser. 


PLATE  2. 


MODES  OF  BACTERIAL  ACTION  25 

instruments;  (d)  Koch's  steam  steriliser,  by  means  of  which  a 
crate  is  slung  in  a  metal  cylinder,  at  the  bottom  of  which  water  is 
boiled;  (e)  the  autoclave,  which  is  the  most  rapid  and  effective 
of  all  the  methods.  This  is  in  reality  a  Koch  steriliser,  but  with 
apparatus  for  obtaining  high  pressure.  The  last  two  (d,  e}  are  used 
for  sterilising  the  nutrient  media  upon  which  bacteria  are  culti- 
vated outside  the  body.  Blood  serum  would,  however,  coagulate 
at  a  temperature  over  60°  C.  (124°  F.),  and  hence  a  special  steriliser 
has  been  designed  to  carry  out  fractional  sterilisation  daily  for  a 
week  at  about  55°  C.-580  C. 


Modes  of  Bacterial  Action 

In  considering  the  specific  action  of  micro-organisms,  it  is  desir- 
able, in  the  first  place,  to  remember  the  two  great  functional  divisions 
of  saprophyte  and  parasite.  A  saprophyte  is  an  organism  that 
obtains  its  nutrition  from  dead  organic  matter.  Its  services,  of 
whatever  nature,  lie  outside  the  tissues  of  living  animals.  Its  life 
is  spent  apart  from  a  "  host."  A  parasite,  on  the  other  hand,  lives 
always  at  the  expense  of  some  other  organism  which  is  its  host,  in 
which  it  lives  or  upon  which  it  lives.  There  is  a  third  or  inter- 
mediate group,  known  as  "  facultative,"  owing  to  their  ability  to  act 
as  parasites  or  saprophytes,  as  the  exigencies  of  their  life  may 
demand. 

The  saprophytic  organisms  are,  generally  speaking,  those  which 
contribute  most  to  the  benefit  of  man,  and  the  parasitic  the  reverse, 
though  this  statement  is  only  approximately  true.  In  their  relation 
to  the  processes  of  fermentation,  decomposition,  nitrification,  etc.,  we 
shall  see  how  great  and  invaluable  is  the  work  which  saprophytic 
microbes  perform.  Their  result  depends,  in  nearly  all  cases,  upon  the 
organic  chemical  constitution  of  the  substances  upon  which  they  are 
exerting  their  action,  as  well  as  upon  the  varieties  of  bacteria  them- 
selves. Nor  must  it  be  understood  that  the  action  of  saprophytes  is 
wholly  that  of  breaking  down  and  decomposition.  As  a  matter  of 
fact,  some  of  their  work  is,  as  we  shall  see,  of  a  constructive  nature  ; 
but,  of  whichever  kind  it  is,  the  result  depends  upon  the  organism  and 
its  environment.  This,  too,  may  be  said  of  the  pathogenic  species, 
all  of  which  are  in  a  greater  or  less  degree  parasitic.  It  is  well 
known  how  various  are  the  constitutions  of  man,  how  the  bodies  of 
some  persons  are  more  resistant  than  those  of  others,  and  how  the 
invading  microbe  will  meet  with  a  different  reception  according  to  the 
constitution  and  idiosyncrasy  of  the  body  which  it  attacks.  Indeed, 
even  after  invasion  the  infectivity  of  the  special  disease,  whatever  it 
happens  to  be,  will  be  materially  modified  by  the  tissues.  When  we 
come  to  turn  to  the  micro-organisms  which  are  pathogenic  parasites 


or  THE 

If  klf  WET*** 


26  THE  BIOLOGY  OF  BACTERIA 

we  shall  further  have  to  keep  clear  in  our  minds  that  their  action  is 
complex,  and  not  simple.  In  the  first  place,  we  have  an  infection  of 
the  body  clue  to  the  bacteria  themselves.  It  may  be  a  general  and 
widespread  infection,  as  in  anthrax,  where  the  bacilli  pass,  in  the 
blood  or  lymph  current,  to  each  and  every  part  of  the  body ;  or  it 
may  be  a  comparatively  local  one,  as  in  diphtheria,  where  the  invader 
remains  localised  at  the  site  of  entrance.  But,  be  that  as  it  may,  the 
micro-organisms  themselves,  by  their  own  bodily  presence,  set  up 
changes  and  perform  functions  which  may  have  far-reaching  effects, 
It  is  obvious  that  the  wider  the  distribution  the  wider  is  the 
area  of  tissue  change,  and  vice  versd.  Yet  there  is  something  of  far 
greater  importance  than  the  mere  presence  of  bacteria  in  human  or 
animal  tissues,  for  the  secondary  action  of  disease-producing  germs — 
and  possibly  it  is  present  in  other  bacteria — is  due  to  their  poisonous 
products,  or  toxines,  as  they  have  been  termed.  These  may  be  of  the 
nature  of  ferments,  and  they  become  diffused  throughout  the  body, 
whether  the  bacteria  themselves  occur  locally  or  generally.  They 
may  bring  about  very  slight  and  even  imperceptible  changes  during 
the  course  of  the  disease,  or  they  may  kill  the  patient  in  a  few  hours. 
Latterly  bacteriologists  have  come  to  understand  that  it  is  not  so 
much  the  presence  of  organisms  which  is  injurious  to  man  and  other 
animals  as  it  is  their  products,  which  cause  mischief ;  and  the 
amount  of  toxic  product  bears  no  known  proportion  to  the  degree  of 
invasion  by  the  bacteria.  The  various  and  widely  differing  modes  of 
action  in  bacteria  are  therefore  dependent  upon  these  three  elements 
(1)  the  tissues  or  medium,  (2)  the  bacteria  or  agents,  and  (3)  the 
products  of  the  bacteria  or  toxins ;  and  in  all  organismal  processes 
these  three  elements  act  and  react  upon  each  other. 

Seed  and  Soil. — It  is  of  essential  importance  to  the  right  under- 
standing of  the  role  which  bacteria  play  in  the  production  of  disease 
to  give  full  place  to  the  part  taken  by  the  soil  on  which  they  are 
implanted.  Few  ideas  in  bacteriology  are  more  erroneous,  or  likely 
to  lead  to  graver  misconception,  than  to  suppose  that  bacteria 
produce  the  same  effect  under  all  conditions,  and  that  the  human 
tissues  play  a  small  part.  One  might  equally  well  expect  seed  to 
behave  in  the  same  way  in  all  kinds  of  soil.  We  know  that  as  a 
fact,  seeds  only  flourish  under  certain  conditions,  and  that  the  soil 
is  only  second  in  importance  to  the  seed-life  itself.  It  is  somewhat 
the  same  in  the  production  of  disease.  The  early  school  of  pre- 
ventive medicine  declared  for  the  health  of  the  individual  and  laid 
the  emphasis  upon  predisposition ;  the  modern  school  have  declared 
for  the  infecting  agent,  and  have  laid  emphasis  upon  the  bacillus. 
The  truth  is  to  be  found  in  a  right  perception  of  the  action  and 
interaction  of  the  tissues  and  the  bacillus.  B.  diphtheric?  in  one 
person's  throat  (A)  sets  up  diphtheria,  in  another  person's  throat 


SEED  AND  SOIL  27 

(B)  lies  quiescent,  producing  no  apparent  disease.  The  cause  of  this 
extraordinary  fact  may  be  a  question  of  different  virulence  in  the 
two  bacilli,  but  is  much  more  likely  to  be  due  to  the  greater  vigour 
and  power  of  resistance  of  the  mucous  membrane  of  B's  throat. 
Sewer  air,  as  we  shall  see  subsequently,  does  not  contain  many 
bacteria,  and  probably  does  not  frequently  convey  germs  of  disease. 
But  this  does  not  prove  that  the  inhalation  of  sewer  air  will  not 
weaken  the  throat,  and  so  form  a  favourable  nidus  for  organisms 
resting  there,  or  organisms  shortly  to  be  inhaled  from  dust  or  mucous 
particles  from  the  throat  of  a  diseased  person.  Which  is  the  more 
important  preventive  method,  to  maintain  the  resistance  of  the 
individual  or  to  waylay  the  infecting  organism,  is  a  nice  point  we  need 
not  attempt  to  decide.  Obviously,  both  objects  should  be  kept  in 
view.  Phthisis  is  another  example.  Thousands  of  persons  inhale 
the  tubercle  bacillus  who  are  not  attacked  by  the  clinical  disease  of 
consumption.  This  fortunate  result  is  due  to  the  resistant  tissues 
of  the  healthy  lung,  and  the  lesson  to  be  derived  is  to  maintain  such 
resistance  at  its  maximum.  This  evidently  is,  in  part,  the  scientific 
explanation  of  Koch's  dictum,  "  It  is  the  overcrowded  dwellings  of 
the  poor  that  we  have  to  regard  as  the  real  breeding  places  of 
tuberculosis ;  it  is  out  of  them  that  the  disease  always  crops  up  anew, 
and  it  is  to  the  abolition  of  these  conditions  that  we  must  first  and 
foremost  direct  our  attention  if  we  wish  to  attack  the  evil  at  its  root 
and  to  wage  war  against  it  with  effective  weapons."*  Part  of  the 
explanation  of  these  words  is  doubtless  that  it  is  in  such  places  that 
the  tubercle  bacillus  breeds  and  passes  from  one  person  to  another. 
But  every  sanitarian  knows  that  the  effect  of  such  environment  is 
to  lower  the  natural  resistance,  to  weaken  the  lung,  impoverish  the 
blood,  and  undermine  the  constitution,  and  thus  a  suitable  nidus  is 
supplied  to  the  invading  bacillus.  "A  perfectly  healthy  lung  is 
seldom  if  ever  primarily  infected  with  the  tubercle  bacillus  "  (Wood- 
head). 

But  the  evidence  of  bacteriology  as  to  the  part  played  by  the  soil 
is  even  stronger  than  at  first  sight  appears.  For  we  now  know,  by 
experiment,  that  micro-organisms  which  in  some  animals  produce 
acute  disease  rapidly  ending  in  death,  result  only  in  mild  disease  in 
other  animals,  and  in  yet  a  third  group  produce  no  apparent  disease 
whatever.  This  is  not  due  to  variation  in  virulence  but  to  variation 
in  soil. 

The  advance  of  bacteriology  has  been  so  rapid  and  marked  by 
such  striking  discoveries  that  there  has  been  a  tendency  to  over-rate 
altogether  the  potentiality  of  the  bacillus  apart  from  its  medium. 
The  latest  findings  in  the  study  of  comparative  culture  work,  of 
immunity  and  of  the  production  of  antitoxins  have,  however,  demon- 

*  Trans.  Brit.  Cong,  of  Tuberculosis,  1901,  vol.  i.,  p.  31. 


28  THE  BIOLOGY  OF  BACTERIA 

strated  beyond  all  doubt  the  enormous  part  played  by  the  medium 
or  soil  in  which  the  micro-organism  is  growing.* 

Specificity  of  Bacteria 

A  species  may  be  denned  as  a  group  of  individuals  which,  however 
many  characters  they  share  with  other  individuals,  agree  in  present- 
ing one  or  more  characters  of  a  peculiar  and  hereditary  kind  with 
some  certain  degree  of  distinctness.-)-  There  is  no  doubt  that  separate 
species  of  bacteria  occur  and  tend  to  remain  as  separate  species. 
Bub  it  must  be  remembered  that  species  are  merely  arbitrary  divisions 
which  present  no  deeper  significance  from  a  philosophical  point  of 
view  than  is  presented  by  well-marked  varieties,  out  of  which 
they  are  in  all  cases  believed  to  have  arisen,  and  from  which  it  is 
often  a  matter  of  individual  opinion  whether  they  shall  be 
separated  by  receiving  a  specific  label.  What  degree  or  character 
of  variation  shall  be  considered  as  sufficient  for  the  demarcation  of 
a  species  of  bacteria  ?  B.  coli  and  B.  typliosus  have  certain  distinctive 
features,  which  are  accepted  as  factors  of  provisional  differentiation. 
But  they  have  many  points  in  common,  the  peculiarity  and  heredity 
of  which  are  not  as  yet  determined.  And  they  have  many  allies, 
para-typhoid  and  para-colon  organisms,  in  the  same  way  as  the 
tubercle  bacillus  possesses  many  allies,  both  bovine  and  human,  among 
acid-fast  species  having  similar  characters  but  differing  in  degree  of 
virulence.  The  fact  is,  that  our  present  knowledge  of  these  matters 
is  very  small,  and  it  is  impossible  to  dogmatise.  The  future  may 
reveal  some  unlooked-for  relationships,  and  organisms  now  classified 
as  morphologically  separate  may  ultimately  prove  to  be  nearly 
related.  Further,  it  may  be  found  that  their  respective  action 
in  the  human  body  is  not  greatly  dissimilar  (the  production, 
of  diarrhoea,  for  example,  by  the  colon  group).  Medium  and 
tissue  have  their  effect  in  the  production  of  variations  of  greater  or 
lesser  mark  in  bacteria.  B.  typhosus  may,  in  the  course  of  sub- 
culture, become  morphologically  indistinguishable  from  B.  coli,  and 
its  pathogenicity  may  also  be  reduced.  The  tubercle  bacillus  in  old 
culture  and  in  saprophytic  existence  becomes  almost  indistinguish- 
able from  the  streptothrix  family.  Streptococcus  conglomeratus  on 
certain  media  simulates  in  a  marked  degree  the  Klebs-Loffler  diph- 
theria bacillus,  and  by  passage  through  a  mouse  loses  its  streptococcal 

*  The  writer  has  been  impressed  in  particular  as  to  the  truth  of  this  view  by 
observation  of  a  number  of  epidemics,  by  the  study  of  a  long  series  of  cultures  of 
the  same  bacillus  on  different  media,  and  by  antitoxin  production.  But  the  same 
conclusion  has  been  reached  from  other  premises.  See  a  suggestive  paper  by  Sir 
W.  J.  Collins,  M.D.,  in  the  Jour,  of  the  Sanitary  Institute  1902  (Oct.),  xxiii.,  pt.  iii., 
p.  335. 

f  Darwin  and  After  Darwin,  G.  J.  Romanes,  F.R.S.,  vol.  ii.,  p.  231. 


ASSOCIATION  OF  ORGANISMS  29 

form  (Gordon).  The  Klebs-Lofiler  bacillus  in  its  turn  may  be 
greatly  modified  in  morphology  and  pathogenicity  by  environment. 
Nor  is  the  change  necessarily  in  descending  order.  Non-pathogenic 
organisms  may  possibly  become  pathogenic.  We  do  not  know. 
The  subject  is  one  full  of  difficulty  in  a  transition  period  of  knowledge 
in  any  branch  of  science.  But  there  is  no  reason  to  suppose  that 
bacteria  are  exceptional  in  nature  and  outside  the  influence  of 
natural  selection ;  and  it  is  not  improbable  that  the  views  of  the  early 
bacteriologists  will  have  to  be  very  much  revised,  and  that  eventually 
it  will  be  found  that  many  "species"  of  micro-organisms  are  in 
reali ty  varieties  of  a  single  species  showing  involution  and  pleomorphic 
forms.  At  the  same  time  it  should  be  recognised  that  amongst  the 
lowliest  forms  of  life  specific  distinctions  are,  as  a  rule,  less  definite, 
and  less  permanent,  than  amongst  forms  of  life  much  higher  in  the 
organic  scale. 

The  Association  of  Organisms 

At  a  later  stage  we  shall  have  an  opportunity  of  discussing 
Symbiosis  and  allied  conditions.  Here  it  is  only  necessary  to  draw 
attention  to  a  fact  that  is  rapidly  becoming  of  the  first  importance 
in  bacteriology.  When  species  were  first  isolated  in  pure  culture  it 
was  found  that  they  behaved  very  differently  under  varying 
circumstances.  This  modification  in  function  has  been  attributed  to 
differences  of  environment  and  physical  conditions.  Whilst  it  is  true 
that  such  external  conditions  must  have  a  marked  effect  upon  such 
sensitive  units  of  protoplasm  as  bacteria,  it  has  recently  been 
proved  that  one  great  reason  why  modification  occurs  in  pure 
artificial  cultures  is  that  the  species  has  been  isolated  from  amongst 
its  colleagues  and  doomed  to  a  separate  existence.  One  of  the  most 
abstruse  problems  in  the  immediate  future  of  the  science  of  bacteri- 
ology is  to  learn  what  intrinsic  characters  there  are  in  species  or 
individuals  which  act  as  a  basis  for  the  association  of  organisms  for  a 
specific  purpose.  Some  bacteria  appear  to  be  unable  to  perform  their 
ordinary  role  without  the  aid  of  others.*  An  example  of  such 
association  is  well  illustrated  in  the  case  of  Tetanus,  for  it  has  been 
shown  that  if  the  bacilli  and  spores  of  tetanus  alone  obtain  entrance 
to  a  wound  the  disease  does  not  follow  the  same  course  as  when  with 
the  specific  organism  the  lactic  acid  bacillus  or  the  common 
organisms  of  suppuration  or  putrefaction  also  gain  entrance.  There 
is  here  evidently  something  gained  by  association.  Again  the  viru- 

*  The  three  different  degrees  of  association  have  been  expressed  by  the  following 
terms  :  Symbiosis,  the  co-operation  for  a  mutual  advantage,  not  obtained  other- 
wise ;  metabiosis,  where  one  organism  prepares  the  way  for  another ;  antibiosis 
(antagonism  of  bacteria),  where  one  of  the  two  associated  organisms  is  directly  or 
indirectly  injuring  the  other. 


30  THE  BIOLOGY  OF  BACTERIA 

lence  of  other  bacteria  is  also  increased  by  means  of  association. 
The  Bacillus  coli  is  an  example,  for,  in  conjunction  with  other 
organisms,  this  bacillus,  although  normally  present  in  health  in  the 
alimentary  canal,  is  able  to  set  up  acute  intestinal  irritation,  and 
various  changes  in  the  body  of  an  inflammatory  nature.  It  is  not 
yet  possible  to  say  in  what  way  or  to  what  degree  the  association  of 
bacteria  influences  their  role.  That  is  a  problem  for  the  future.  But 
whilst  we  have  examples  of  this  association  in  Streptococcus  and  the 
bacillus  of  diphtheria,  B.  coli  and  yeasts,  Tetanus  and  putrefactive 
bacteria,  Diplococcus  pneumonice  and  Proteus  vulgaris,  and  Streptococcus 
erysipelatis  and  Proteus  vulgaris,  we  cannot  doubt  that  there  is  an 
explanation  to  be  found  of  many,  hitherto  unknown,  results  of 
bacterial  action.  This  is  the  place  in  which  mention  should  also  be 
made  of  higher  organisms  associated  for  a  specific  purpose  with 
bacteria.  There  is  some  evidence  to  support  the  belief  that  some  of 
the  Leptotrichese  (Crenothrix,  Beggiatoa,  Leptothrix,  etc.)  and  the 
Cladotricheae  (Cladothrix)  perform  a  preliminary  disintegration  of 
organic  matter  before  the  decomposing  bacteria  commence  their 
labours.  This  occurs  apparently  in  the  self-purification  of  rivers,  as 
well  as  in  polluted  soils. 

Antagonism  of  Bacteria  (Antibiosis). — Study  of  the  life- 
history  of  many  of  the  water  bacteria  will  reveal  the  fact  that  they 
can  live  and  multiply  under  conditions  which  would  at  once  prove 
fatal  to  other  species.  Some  of  these  water  organisms  can  indeed 
increase  and  multiply  in  distilled  water,  whereas  it  is  known  that 
other  species  cannot  even  live  in  distilled  water  owing  to  the  lack  of 
pabulum.  Thus  we  see  that  what  is  favourable  for  one  species  may 
be  the  reverse  for  another. 

Further,  we  shall  have  opportunity  of  observing,  when  consider- 
ing the  bacteriology  of  water  and  sewage,  that  there  is  in  these 
media  in  nature  a  keen  struggle  for  the  survival  of  the  fittest 
bacteria  for  each  special  medium.  In  a  carcase  it  is  the  same.  If 
saprophytic  bacteria  are  present  with  pathogenic,  there  is  a  struggle 
for  the  survival  of  the  latter.  Now  whilst  this  is  in  part  due  to  a 
competition  owing  to  a  limited  food  supply  and  an  unlimited  popula- 
tion, as  occurs  in  other  spheres,  it  is  also  due  in  part  to  the  inimical 
influence  of  the  chemical  products  of  the  one  species  upon  the  life  of 
the  bacteria  of  the  other  species.  Moreover,  in  one  culture  medium, 
as  Cast  has  pointed  out,  two  species  will  often  not  grow.  When 
Pasteur  found  that  exposure  to  air  attenuated  his  cultures,  he 
pointed  out  that  it  was  not  the  air  perse  that  hindered  growth,  but  it 
was  the  introduction  of  other  species  which  competed  with  the 
original.  The  growth  of  the  spirillum  of  cholera  is  opposed  by 
Bacillus  pyogcnes  foetidus.  B.  anthracis  is,  in  the  body  of  animals, 
opposed  by  either  B.  pyocyaneus  or  Streptococcus  erysipelatis,  and  yet 


or 

££j 

ANTAGONISM  AND  ATTENUATION  31 

it  is  aided  in  its  growth  by  B.  prodigiosus.  B.  aceti  is  under  certain 
circumstances  antagonistic  to  B.  coli. 

In  several  of  the  reports  of  the  late  Sir  Eichard  Thome  issued 
from  the  Medical  Department  of  the  Local  Government  Board,  we 
have  the  record  of  a  series  of  experiments  performed  by  Dr  Klein 
upon  the  subject  of  the  antagonisms  of  microbes.  From  this  work  it 
is  clearly  demonstrated  that  whatever  opposition  one  species  affords 
to  another  it  is  able  to  exercise  by  means  of  its  poisonous  properties. 
These  are  of  two  kinds.  There  is,  as  is  now  widely  known,  the 
poisonous  product  named  the  toxin,  into  which  we  shall  have  to 
inquire  in  more  detail  at  a  later  stage.  There  is  also  in  many 
species,  as  several  workers  have  pointed  out,  a  poisonous  constituent, 
or  constituents,  included  in  the  body  protoplasm  of  the  bacillus,  and 
which  he  therefore  terms  the  intracellular  poison.  Now,  whilst  the 
former  is  different  in  every  species,  the  latter  may  be  a  property 
common  to  several  species.  Hence  those  having  a  similar  intracellu- 
lar poison  are  antagonistic  to  each  other,  each  member  of  such  a 
group  being  unable  to  live  in  an  environment  of  its  own  intracellular 
poison.  Further,  it  has  been  suggested  that  there  are  organisms 
possessing  only  one  poisonous  property,  namely,  their  toxin — for 
example,  the  bacilli  of  Tetanus  and  Diphtheria — whilst  there  are 
other  species,  as  above,  possessing  a  double  poisonous  property,  an 
intracellular  poison  and  a  toxin.  In  this  latter  class  would  be 
included  the  bacilli  of  Anthrax  and  Tubercle. 

There  can  be  no  doubt  that  these  complex  biological  properties 
of  association  and  antagonism,  as  well  as  the  parasitic  growth  of 
bacteria  upon  higher  vegetables,  are  as  yet  little  understood,  and  we 
may  be  glad  that  any  light  is  being  shed  upon  them.  In  the 
biological  study  of  soil  bacteria  in  particular  may  we  expect  in  the 
future  to  find  examples  of  association,  even  as  already  there  are  signs 
that  this  is  so  in  certain  pathogenic  conditions.  In  the  alimentary 
canal,  on  the  other  hand,  and  in  conditions  where  organic  matter  is 
greatly  predominating,  we  may  expect  to  see  further  light  on  the 
subject  of  antagonism. 

Attenuation  of  Virulence  or  Function. — It  was  pointed  out 
by  some  of  the  pioneer  bacteriologists  that  the  function  of  bacteria 
suffered  under  certain  circumstances  a  marked  diminution  in  power. 
Later  workers  found  that  such  a  change  might  be  artificially  pro- 
duced. Pasteur  introduced  the  first  method,  which  was  the  simple 
one  of  allowing  cultures  to  grow  old  before  sub-culturing.  Obviously 
a  pure  culture  cannot  last  for  ever.  To  maintain  the  species  in 
characteristic  condition  it  is  necessary  frequently  to  sub-culture  upon 
fresh  media.  If  this  simple  operation  be  postponed  as  long  as 
possible  consistent  with  vitality  and  then  performed,  it  will  be  found 
that  the  sub-culture  is  attenuated,  i.e.,  weakened.  Another  mode  is 


32  THE  BIOLOGY  OF  BACTERIA 

to  raise  the  pure  culture  to  a  temperature  approaching  its  thermal 
death-point.  A  third  way  of  securing  the  same  end  is  to  place  it 
under  disadvantageous  external  circumstances,  for  example  in  a  too 
alkaline  or  too  acid  medium.  A  fourth  method  is  to  pass  it  through 
the  tissues  of  an  insusceptible  animal.  Thus  we  see  that,  whilst 
the  favourable  conditions  which  we  have  considered  afford  full 
scope  for  the  growth  and  performance  of  functions  of  bacteria,  we  are 
able  .by  a  partial  withdrawal  of  these,  short  of  that  ending  fatally, 
to  modify  the  character  and  strength  of  bacteria.  In  future  chapters 
we  shall  have  opportunity  of  observing  what  can  be  done  in  this 
direction. 

Bacterial  Diseases  of  Plants 

Eeference  has  been  made  to  the  associated  work  of  higher 
vegetable  life  and  bacteria.  The  converse  is  also  true.  Just  as 
we  have  bacterial  diseases  affecting  man  and  animals,  so  also  plant 
life  has  its  bacterial  diseases.  "Wakker,  Prillieux,  Erwin  Smith, 
and  others  have  investigated  the  pathogenic  conditions  of  plants 
due  to  bacteria,  and  though  this  branch  of  the  science  is  in  its 
very  early  stages,  many  facts  have  been  learned.  Hyacinth  disease 
is  due  to  a  flagellated  bacillus.  The  wilt  of  cucumbers  and  pumpkins 
is  a  common  disease  in  some  districts  of  the  world,  and  may 
cause  widespread  injury.  It  is  caused  by  a  micro-organism 
which  fills  the  water-ducts.  Wilting  vines  are  full  of  the  same 
sticky  germs.  Desiccation  and  sunlight  have  a  strong  prejudicial 
effect  upon  these  organisms.  Melon  Uight  must  not  be  confused 
with  the  bacterial  wilt  of  cucumbers  and  melons.  The  blight  disease 
is  caused  by  Plasmopara  cubensis,  a  sporulating  fungus.  Bacterial 
brown-rot  of  potatoes  and  tomatoes  is  another  plant  disease  probably 
due  to  a  bacillus.  The  bacillus  passes  down  the  interior  of  the 
stem  into  the  tubers,  and  brown-rots  them  from  within.  There  is 
another  form  of  brown-rot  which  affects  cabbages.  It  blackens  the 
veins  of  the  leaves,  and  a  woody  ring  which  is  formed  in  the  stem 
causes  the  leaves  to  fall  off.  This  also  is  due  to  a  micro-organism, 
which  gains  entrance  through  the  water-pores  of  the  leaf,  and 
subsequently  passes  into  the  vessels  of  the  plants.  It  multiplies 
by  simple  fission,  and  possesses  a  flagellum.  Certain  diseases  of 
Sweet  Corn  have  been  investigated  by  Stewart,  and  traced  to  a 
causal  bacillus  possessing  marked  characters.  Professor  Potter 
believes  that  white-rot  of  the  turnip  is  produced  by  Pseudomonas 
destructans,  a  liquefying,  motile,  aerobic  bacillus. 


CHAPTEE  II 

BACTERIA   IN   WATER 

Quantity  of  Bacteria  in  Water— Quality  of  Water  Bacteria :  (a)  Ordinary  Water 
Bacteria ;  (6)  Sewage  Bacteria  ;  B.  coll  communis  ;  (c)  Pathogenic  Bacteria  in 
Water — Interpretation  of  the  Findings  of  Bacteriology — Natural  Purification 
of  Water— Artificial  Purification  of  Water— Sand  Filtration— Domestic  Puri- 
fication of  Water. 

The  collection  of  samples,  though  it  appears  simple  enough,  is 
sometimes  a  difficult  and  responsible  undertaking.  Complicated 
apparatus  is  rarely  necessary,  and  fallacies  will  generally  be  avoided 
by  observing  two  directions.  In  the  first  place,  the  sample  should 
be  chosen  as  representative  as  possible  of  the  real  water  or  conditions 
we  wish  to  examine.  Some  authorities  advise  that  it  is  necessary 
to  allow  the  tap  to  run  for  some  minutes  previously  to  collecting 
the  sample;  but  if  we  desire  to  examine  chemically  for  lead  or 
biologically  for  micro-organisms  in  the  pipes,  then  such  a  proceeding 
would  be  injudicious.*  If  it  is  well  water  that  is  to  be  examined, 
the  well  should  be  pumped  for  some  minutes  before  taking  the 
sample.  If  it  is  river  water  which  is  to  be  examined,  it  is  important 
to  collect  the  sample  without  incorporating  any  deposit.  In  short, 
we  must  use  common  sense  in  the  selection  and  obtaining  of  a 
sample,  following  this  one  guide,  namely,  to  collect  as  nearly  as 
possible  a  sample  of  the  exact  water,  the  quality  of  which  it  is 
desired  to  learn.  In  the  second  place,  we  must  observe  strict 

*  Water  from  a  house  cistern  is  rarely  a  fair  sample  of  a  town  supply.  It 
should  be  taken  from  the  main.  If  taken  from  a  stream  or  still  water,  the  collect- 
ing bottle  should  be  held  about  a  foot  below  the  surface  before  the  stopper  is 
removed. 

33 


34  BACTERIA  IN  WATER 

bacteriological  cleanliness  in  all  our  manipulations.  This  means  that 
we  must  use  only  sterilised  vessels  or  flasks  for  collecting  the  sample, 
and  in  the  manipulation  required  we  must  be  extremely  careful  to 
avoid  any  pollution  of  air  or  any  addition  to  the  organisms  of  the 
water  from  unsterilised  apparatus.  A  flask  polluted  in  only  the 
most  infinitesimal  degree  will  entirely  vitiate  all  results.  Vessels 
may  be  sterilised  by  heating  at  150°  C.  for  two  or  three  hours.  If 
this  is  impracticable  the  vessel  may  be  washed  with  pure  sulphuric 
acid,  and  then  thoroughly  rinsed  out  in  the  water  which  is  to  be 
examined. 

Accompanying  the  sample  should  be  a  more  or  less  full  statement 
of  its  source.  There  can  be  no  doubt  that,  in  addition  to  a  chemical 
and  bacteriological  report  of  a  water,  there  should  also  be  made  a 
careful  examination  of  its  source.  This  may  appear  to  take  the 
bacteriologist  far  afield,  but  until  he  has  seen  for  himself  what  "  the 
gathering  ground "  is  like,  and  from  what  sources  come  the  feeding 
streams,  he  cannot  judge  the  water  as  fairly  as  he  should  be  able  to 
do.  The  configuration  of  the  gathering  ground,  its  subsoil,  its 
geology,  its  rainfall,  its  relation  to  the  slopes  which  it  drains,  the 
nature  of  its  surface,  the  course  of  its  feeders,  and  the  absence  or 
presence  of  cultivated  areas,  of  roads,  of  houses,  of  farms,  of  human 
traffic,  of  cattle  and  sheep — all  these  points  should  be  noted,  and 
their  influence,  direct  or  indirect  upon  the  water,  carefully  borne  in 
mind. 

When  the  sample  has  been  duly  collected,  sealed,  and  a  label 
affixed  bearing  the  date,  time,  and  conditions  of  collection  and  full 
address,  it  should  be  transmitted  with  the  least  possible  delay  to  the 
laboratory.  Frequently  it  is  desirable  to  pack  the  bottles  in  a  small 
ice-case  for  transit.  Miquel,  Pakes,  and  others  have  constructed 
special  forms  of  packing-cases,  and  these  have  their  advantages. 
But  the  ordinary  bottle  of  water  may  be  quite  satisfactorily  con- 
veyed, as  a  rule,  packed  in  sawdust  and  ice.  On  receipt  of  such  a 
sample  of  water  the  examination  must  be  immediately  proceeded 
with,  in  order  to  avoid,  as  far  as  possible,  the  fallacies  arising  from 
the  rapid  multiplication  of  germs. 

Multiplication  of  Bacteria  in  Water. — In  almost  pure  water,  at  the 
ordinary  temperature  of  a  room,  Frankland  found  that  organisms 
multiplied  as  follows : 

No.  of  Germs. 
Hours.  per  c.c. 

0  .             .            •«  1,073 

6  ...  6,028 

24  .  .  *  7,262 

48  .  »  .  48,100 

Another  series  of  observations  revealed  the  same  sort  of  rapid 


MULTIPLICATION  OF  WATER  BACTERIA  35 

increase  of  bacteria.  On  the  date  of  collection  the  micro-organisms 
per  c.c.  in  a  deep-well  water  (in  April)  were  seven.  After  one  day's 
standing  at  room  temperature  the  number  had  reached  twenty-one 
per  c.c.  After  three  days  under  the  same  conditions  it  was  495,000 
per  c.c.  At  blood-heat  the  increase  would,  of  course,  be  much  greater, 
as  a  higher  temperature  is  more  favourable  to  multiplication.  But 
this  would  depend  in  part  also  upon  the  degree  of  impurity  in  the 
water,  a  pure  water  decreasing  in  number  of  germs  on  account  of  the 
exhaustion  of  the  pabulum,  whereas,  for  the  first  few  days  at  all 
events,  an  organically  polluted  water  would  show  an  enormous 
increase  in  bacteria. 

It  is  desirable  to  remember  that  organisms,  in  an  ordinary  water, 
do  not  continue  to  increase  indefinitely.  Cramer,  of  Zurich,  examined 
the  water  of  the  Lake  after  it  had  been  standing  in  a  vessel  for 
different  periods,  with  the  following  results: — 

Hours  and  Days  of  No.  of  Micro-organisms 

Examination.  per  c.c. 

0  hours  143 


24   „ 
3  days 

8  ,, 
17  „ 
70  „ 


12,457 
328,543 
233,452 

17,436 
2,500 


In  a  general  way  it  may  be  said  that  foul  waters,  rich  in  putrescible 
animal  matter,  show  a  rapid  increase  of  bacteria ;  surface  waters,  such 
as  river  water,  show  a  slow  and  persistent  multiplication  of  organisms ; 
and  deep -well  waters  and  spring  water  show  comparatively  little 
increase  in  contained  bacteria.  Indeed  it  may  be  said  that  the 
condition  of  a  water  is  partly  indicated  by  the  rapidity  or  slowness 
with  which  its  bacteria  increase.  A  low  temperature  (5°  C.) 
undoubtedly  diminishes  the  multiplication,  and  there  are  other 
conditions  such  as  exposure  to  air,  movement,  and  antagonism  of 
organisms  which  exert  an  indirect  effect.  As  will  be  inferred  from 
what  has  been  said,  the  most  important  condition  affecting  the 
number  of  bacteria  in  a  water  is  the  organic  matter  contained  in  it.* 


-The  Bacteriology  of  Water 

In  many  natural  waters  there  will  be  found  varied  contents  even 
in  regard  to  flora  alone:    algce,  diatoms,  spirogyrce,  desmids,  and  all 

*  For  suggestions  and  hints  on  points  of  technique  in  the  systematic  examination 
of  a  water,  see  Delepine's  Bacteriological  Survey  of  Surface  Water  Supplies  :  Jour,  of 
State  Medicine,  1898,  vol.  vi.,  pp.  145,  193,  241,  289  ;  and  Bacteriological  Examina- 
tion of  Water,  by  W.  H.  Horrocks.  (See  also  present  volume,  pp.  463-473.) 


36  BACTERIA  IN  WATER 

sorts  of  vegetable  detritus.  Many  of  these  organisms  are  held 
responsible  for  certain  disagreeable  tastes  and  odours.  The  colour 
of  a  water  may  also  be  due  to  similar  causes.  Dr  Garrett,  of 
Cheltenham,  has  recorded  the  occurrence  of  redness  of  water  owing 
to  a  growth  of  Crenothrix  polyspora,  and  many  other  similar  cases 
make  it  evident  that  not  unfrequently  great  changes  may  be  produced 
in  water  by  contained  microscopic  vegetation. 

With  the  exception  of  water  from  springs  and  deep  wells,  all 
unfiltered  natural  waters  contain  numbers  of  bacteria.*  The  actual 
number  roughly  depends,  as  we  have  seen,  upon  the  amount  of 
organic  pabulum  present,  and  upon  certain  physical  conditions  of 
the  water.  In  some  species  multiplication  does  not  appear  to 
depend  on  the  presence  of  much  organic  matter,  and,  indeed,  sonle 
bacteria  can  live  and  multiply  in  almost  pure  water ;  e.g.,  Microeoccus 
aquatilis  and  Bacillus  erythrosporus.  Again,  others  depend  not  upon 
the  quantity  of  organic  matter,  but  upon  its  quality.  And  frequently 
in  a  water  of  a  high  degree  of  organic  pollution  it  will  be  found  that 
bacteria  have  been  restrained  in  their  development  by  the  competi- 
tion of  other  species  monopolising  the  pabulum.  It  will  be  necessary 
to  deal  with  the  subject  under  the  two  subdivisions  of  (1)  quantity 
and  (2)  quality  of  bacteria  found  in  water. 


Quantity  of  Bacteria  in  Water 

Percy  Frankland  has  quoted  in  his  book  •(•  a  number  of  records 
of  the  quantity  of  organisms  found  in  various  waters.  These  tables 

five  the  returns  for  the  rivers  Seine  (Miquel),  Ehone,  Saone  (Koux), 
pree  (Frank),  Isar  (Prausnitz),  Limmat  (Schlatter),  Ehine  (Mcers), 
etc.  Here  it  is  unnecessary  to  do  more  than  give  typical  illustra- 
tions, and  for  comparative  purposes  English  rivers  may  be  taken. 
Prof.  Frankland  himself  collected  water  from  the  river  Thames 
at  various  times  and  seasons,  and  some  of  his  results  were  as 
follow : — 

*  Bacteria,  of  course,  exist  in  the  water  of  the  sea.  Near  land,  as  might  be 
expected,  the  number  is  greatest,  and  diminishes  rapidly  further  out  to  sea. 
Currents  sometimes  bring  them  to  the  surface  from  a  depth  of  596  fathoms 
(Fischer).  At  a  depth  of  100-200  fathoms  bacteria  have  been  found  in  large 
numbers.  The  comparative  paucity  at  the  surface  is  due  to  the  germicidal  effect 
of  sunlight.  Ocean  bacteria  vary  widely -in  size  and  shape.  Apparently,  typical 
cocci  and  bacilli  are  never  met  with  on  the  high  seas.  Spirilla  and  zooglooa 
masses  are  common.  Most  sea  bacteria  are  motile  and  furnished  with  flagella ; 
some  are  anaerobes. 

f  Micro-organisms  in  Water  (1894),  pp.  89-116. 


NUMBER  OF  BACTERIA  IN  WATER 


37 


River  Thames   Water  Collected  at  Hampton. 

Number  of  Micro-organisms  obtained  from  1  c.c.  of  Water. 


Mouth. 
January 
February 
March 
April  . 
May    . 
June    . 
July     . 
August 
September 
October 
November 
December 


45,000 

15,800 

11,415 

12,250 

4,800 

8,300 

3,000 

6,100 

8,400 

8,600 

56,000 

63,000 


1887. 

30,800 

6,700 

30,900 

52,100 

2,100 

2,200 

2,500 

7,200 

16,700 

6,700 

81,000 

19,000 


92,000 

40,000 

66,000 

13,000 

1,900 

3,500 

1,070 

3,000 

1,740 

1,130 

11,700 

10,600 


Another  example  from  the  river  Lea  was  as  follows : — 
River  Lea   Water  Collected  at  Chingford. 


Number  of  Micro-organisms  obtained  from  1  c.c.  of  Water. 


Month. 
January 
February 
March 
April   . 
May    . 
June   . 
July    . 
August 
September 
October 
November 
December 


1886. 

39,300 

20,600 

9;025 

7,300 

2,950 

4,700 

5,400 

4,300 

3,700 

6,400 

12,700 

121,000 


1887. 

37,700 

7,900 

24,000 

1,330 

2,200 

12,200 

12,300 

5,300 

9,200 

7,600 

27,000 

11,000 


31,000 

26,000 

63,000 

84,000 

1,124 

7,000 

•2,190 

2,000 

1,670 

2,310 

57,500 

4,400 


"  During  the  summer  months  these  waters  are  purest  as  regards 
micro-organisms,  this  being  due  to  the  fact  that  during  dry  weather 
these  rivers  are  mainly  composed  of  spring  water,  whilst  at  other 
seasons  they  receive  the  washings  of  much  cultivated  land  "  (Frank- 
land).  Prausnitz  has  shown  that  water  differs,  as  would  be  expected, 
according  to  the  locality  in  the  stream  at  which  examination  is  made. 
His  investigations  were  made  from  the  river  Isar  before  and  after  it 
receives  the  drainage  of  Munich  :— 


Above  Munich 

Near  entrance  of  principal  sewer 

13  kilometres  from  Munich    . 

22 

33 


No.  of  Colonies 
per  c.c. 

531 

227,369 
9,111 

4,796 

2,378 


38 


BACTERIA  IN  WATER 


Frankland  has  shown  that  the  river  Dee  affords  another  example, 
even  more  perfect,  of  pollution  and  restoration  repeated  several  times 
until  the  river  becomes  almost  bacterially  pure. 

Professor  Boyce  and  his  colleagues  have  recently  made  an 
examination  of  the  river  Severn  before  and  after  its  waters  pass  the 
town  of  Shrewsbury.*  Their  findings  may  be  represented  briefly  as 
follows : — 


Average  Total 

Average 

Position  of  Examination. 

No.  of  Bacteria 

No.  of  B.  coli 

per  c.c. 

per  c.c. 

At  Asylum,  2  miles  above  Shrewsbury 
Waterworks,  opposite  Shrewsbury 

7,000 
13,000 

13 

46 

Ferry  i.,  0'6  of  a  mile  lower  down 

20,000 

177 

English  Bridge,  1  '6  of  a  mile  lower  down 

23,000 

321 

Ferry  iii.,  2  '5  miles  lower  down 

19,000 

600 

Uffington,  4*7  miles  lower  down 

17,000 

142 

Alcham,  9  miles  lower  down    . 

13,000 

48 

Cressage,  16  miles  lower  down 

5,000 

36 

This  table  and  that  of  Prausnitz — and  many  other  workers  have 
produced  similar  records — illustrate  the  effect  of  (a)  local  pollution, 
and  (&)  river  purification,  upon  the  bacterial  content  of  water,  to 
which  subsequent  reference  wili  be  made.  The  record  respecting  the 
Severn  includes  also  the  indication  of  sewage  pollution  by  the 
presence  of  B.  coli.  An  elaborate  examination  has  also  been  made 
of  the  water  of  the  river  Thames  and  the  Thames  estuary,  by 
Houston,  and  the  report  dealing  with  it  is  full  of  information  on 
the  subject,  to  which  reference  should  be  made.f 

Lastly,  the  accompanying  table  (pp.  39  and  40),  for  1902  and  1903, 
deals  with  the  London  water  supply  as  examined  by  Crookes  and 
Dewar.  J  It  is  concerned,  it  should  be  added,  only  with  numerical 
results. 

This  record,  compiled  from  the  monthly  reports  respecting  the  three 
waters  supplied  to  the  metropolis,  illustrates  many  interesting  points 
upon  which  we  have  not  space  to  dwell  fully.  A  few  notes,  however, 
upon  an  actual  example  are  more  useful  than  much  theoretical 
information,  and  therefore  a  brief  study  of  these  figures  may  be  made. 
In  the  main  the  table  illustrates  two  points  more  clearly  than  the 
preceding  tables.  The  first  is  the  effect  of  filtration,  and  the  second 
is  the  effect  of  season,  upon  the  number  of  bacteria  in  water.  In 
respect  to  the  former,  comment  is  needless.  It  is  only  necessary  to 

*  Royal  Commission  on  Sewage  Disposal,  Second  Report,  1902,  p.  99. 
t  Ibid.,  Fourth  Report,  1904',  vol.  iii.,  pp.  1-75. 
J  Metropolitan  Water  Supply,  1902  and  1903. 


BACTERIA  IN   LONDON  WATER 


39 


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BACTERIA  IN  LONDON  WATER  41 

examine  the  returns  to  recognise  the  marked  reduction  in  the  number 
of  bacteria,  in  some  cases  amounting  to  98  per  cent.,  brought  about 
by  filtration.  In  respect  to  the  latter,  the  effect  of  season,  some  note 
is  required.  It  will  be  seen  that  during  1902  the  figures  are  fairly 
uniform  throughout  the  year,  showing,  on  the  whole,  a  rise  in  winter 
and  spring,  and  a  fall  during  summer.  But  in  1903  the  returns  show 
wide  variation  which  calls  for  explanation,  which  is  as  follows : — 

The  water  supply  in  December  1902,  on  the  whole,  maintained  an  equal  microbic 
purity  to  that  of  November.  This  exceptional  condition  of  the  supply,  the  com- 
paratively small  number  of  bacteria,  for  the  winter  months  is  no  doubt  due  to  the 
absence  of  floods  in  the  Thames  Valley,  and  to  the  unusually  mild  character  of  the 
season.  "If  the  large  deficit  in  the  rainfall  is  made  up,"  wrote  Crookes  and 
Dewar,  *  *  no  doubt  there  will  be  in  the  near  future  a  period  when  the  filtration  of 
the  London  waters  will  require  more  than  usual  care.  As  the  general  filter-beds 
are,  however,  in  good  working  order,  we  believe  these  difficulties,  should  they 
arise,  will  be  overcome  satisfactorily." 

The  standard  of  general  organic  purity  during  1902,  as  defined  by  chemical 
methods,  was  maintained.  As  regards  the  month  of  December,  the  Thames-derived 
Companies  showed  decided  differences  among  themselves,  which,  as  the  supply 
comes  from  the  same  source,  were  essentially  connected  with  differences  in  storage 
capacity  and  variations  in  the  structure  of  the  filter-beds,  although  the  latter  is  of 
less  importance  in  removing  soluble  organic  matter. 

Crookes  and  Dewar  add:  "The  longer  our  experience  of  the  bacteriological 
method,  as  applied  to  the  analysis  of  the  filtered  supply,  and  the  wider  its  application, 
the  more  we  are  convinced  of  its  primary  importance  as  a  safeguard  to  the  public. 
It  enables  us  to  define  in  a  much  more  delicate  way  than  is  possible  by  chemical 
analysis  what  is  an  efficiently  filtered  water,  and  thereby  enables  the  chemist  to 
warn  the  engineer  the  moment  any  one  of  his  filters  show  signs  of  defective 
working.  Whether  the  supply  as  regards  the  organic  matter  in  solution  varies 
more  or  less  according  to  the  season  of  the  year,  is  of  relatively  small  moment 
as  compared  with  the  knowledge  that  the  microbic  impurity  is  reduced  to  a 
minimum. " 

That  was  the  position  at  the  end  of  1902.  But  at  the  turn  of  the  year,  owing  to 
the  great  increase  in  the  rainfall,  the  microbes  in  the  unfiltered  Thames  water  rose 
from  about  6000  to  13,000,  that  is,  the  bacteriological  impurity  about  doubled, 
whereas  the  unfiltered  New  River  water  underwent  little  or  no  alteration.  The 
result  of  this  increase  was  that  the  filters  of  the  Thames-derived  Companies,  which 
were  not  working  at  their  best,  furnished  a  larger  number  of  samples  from  the  filter 
wells,  showing  an  increase  in  the  number  of  bacteria  which  was  the  inevitable  result 
of  an  increased  rainfall. 

Things  remained  thus  until  April,  when  in  comparison  with  the  month  of  March 
the  bacteriological  quality  showed  considerable  improvement,  a  result  which  might 
have  been  anticipated  from  the  advent  of  summer,  and  the  improved  natural 
conditions  associated  with  vegetable  growth ;  a  state  of  things  which  generally 
improves  the  quality  of  the  water  obtained  from  such  collecting  areas  as  the  valleys 
of  the  Thames  and  Lea.  But  in  June,  when  the  number  of  bacteria  ought  to  have 
been  low,  as  ordinarily  there  would  be  a  small  rainfall,  an  exceptional  condition  of 
things  arose.  The  total  excess  of  rainfall  amounted  to  49 '9  per  cent,  on  the  thirty 
years'  average,  so  that  during  the  month  of  June  actually  22 -5  per  cent,  or  an 
amount  approaching  one-half  of  the  previous  excess,  of  rain  fell  in  the  valley  of  the 
Thames.  Such  an  amount  of  rain  is  altogether  exceptional  in  twenty- two  years' 
experience.  The  result  was  that  the  proportion  of  vegetable  matter  in  solution, 
and  therefore  the  colour  of  the  water,  were  both  quite  exceptional  for  the  summer 
months.  Nevertheless,  the  general  filtration  was  adequately  and  effectively 
performed,  as  is  shown  by  the  bacteriological  results.  Similar  conditions  prevailed 
in  August  and  October.  The  exceptional  rainfall,  which  amounted  to  sixty  per 


43  BACTERIA  IN  WATER 

cent,  in  excess  of  the  average,  kept  the  colour  and  amount  of  soluble  vegetable 
matter  in  solution  abnormally  high.  In  December,  owing  to  the  continued  rains,  the 
New  River  and  Thames  unfiltered  waters  contained  a  maximum  number  of  bacteria. 
In  dry  weather  the  number  per  c.c.  had  been  as  low  as  149  and  2013  respectively, 
but  owing  to  seasonal  changes  they  had  risen  to  861  and  27,216  bacteria  per  c.c. 
respectively. 

From  these  various  records  we  find  that  in  the  result  the  number 
of  bacteria  in  river  water  depends  upon  a  variety  of  circumstances, 
amongst  which  the  most  important  direct  conditions  are  four,  namely, 
(1)  local  pollution,  (2)  natural  purification  (to  which  subsequent 
reference  will  be  made),  (3)  season  and  rainfall,  and  (4)  sedimentation 
and  filtration.  Behind  these  direct  conditions  we  have  also  seen  that 
time,  temperature,  light,  exposure  to  air,  and  the  presence  of  organic 
matter  play  an  essential  part. 

Bacteriological  Examination  of  Water. — [See  Appendix,  p.  463.] 

Quantitative  Standard. — In  arriving  at  a  conclusion  respecting  the 
number  of  organisms  in  a  water  and  their  bearing  upon  its  suitability 
for  use,  it  should  be  remembered  that  a  chemical  report  and  a 
bacteriological  report  are  desirable  before  forming  an  opinion.  The 
former  is  able  to  tell  us  the  quantity  of  salts  and  condition  of  the 
organic  matter  present :  the  latter  the  number  and  quality  of  micro- 
organisms. Neither  can  take  the  place  of  the  other,  and,  generally 
speaking,  both  are  more  or  less  useless  until  we  can  learn,  by  inspec- 
tion and  investigation  of  the  source  of  the  water,  the  origin  of  the 
organic  matter  or  contamination.  Hence  a  water  report  should  con- 
tain not  only  a  record  of  physical  and  microscopical  characters,  of 
chemical  constituents,  and  of  the  presence  or  absence  of  micro- 
organisms, injurious  and  otherwise,  but  it  should  also  contain  infor- 
mation obtained  by  personal  investigation  of  the  source.  Only  thus 
can  a  reasonable  opinion  be  expected.  Moreover,  it  is  generally  only 
possible  to  form  an  accurate  judgment  of  a  water  by  watching  its 
history ;  that  is  to  say,  not  from  one  examination  only,  but  from  a 
series  of  observations.  The  writer  has  examined  a  certain  water 
supply  for  thirty-six  consecutive  months.  In  1901  the  average 
number  of  bacteria  per  c.c.  was  93,  in  1902,  136,  and  in  1903,  57. 
This  shows  a  stable  bacterial  content  which  in  itself  is  favourable. 
A  water  yielding  a  steady  standard  of  bacterial  content  is  a  much 
more  satisfactory  water,  from  every  point  of  view,  than  one  which 
is  unstable,  one  month  possessing  50  bacteria  per  c.c.  and  another 
month  5000.  It  is  obvious  that  rainfall  and  drought,  soil  and  trade 
effluents,  time  and  temperature,  will  have  their  influence  in  materially 
affecting  the  bacterial  condition  of  a  water. 

Miquel  and  others  have  suggested  standards  which  allow  "  very 
pure  water"  to  contain  up  to  100  micro-organisms  per  c.c.  Pure 
water  must  not  contain  more  than  1000,  and  water  containing  up  to 


NUMERICAL  STANDARDS  43 

100,000   bacteria  per  c.c.   is   contaminated   with   surface  water  or 
sewage.     Mace  gives  the  following  table : — 

Bacteria  per  c.c. 

Very  pure  water     .  .  0       to  50 

Good  water  .          -  .  50       „  500 

Passable  (mediocre)  water  500       „        3,000 

Bad  water.  .  »'        3,000       ,,       10,000 

Very  bad  water       .  k         10,000       „     100,000  and  over. 

Koch  first  laid  emphasis  on  the  quantity  of  bacteria  present  as  an 
index  of  pollution,  and  whilst  different  authorities  have  all  agreed 
that  there  is  a  necessary  quantitative  limit,  it  has  been  impossible  to 
arrive  at  a  settled  standard  of  permissible  impurity.  Besson  adopts 
the  standard  suggested  by  Miquel,  and  on  the  whole  French 
bacteriologists  follow  suit.  They  also  agree  with  him,  generally 
speaking,  in  not  placing  much  emphasis  upon  the  numerical  estima- 
tion of  bacteria  in  water.  In  Germany  and  England  it  is  the  custom 
to  adopt  a  stricter  limit.  Koch  in  1893  suggested  100  bacteria 
per  c.c.  as  the  maximum  number  of  bacteria  which  should  be  present 
in  a  properly  filtered  water.  Miquel  holds  that  not  more  than  ten 
different  species  of  bacteria  should  be  present  in  a  drinking  water, 
and  such  is  a  useful  standard.  The  presence  of  many  rapidly 
liquefying  bacteria  or  organisms  associated  with  sewage  or  surface 
pollution  would,  even  though  present  in  fewer  numbers  than  a 
standard,  condemn  a  water.  From  a  consideration  of  all  the  facts 
it  will  be  seen  that  it  is  impossible  to  judge  alone  by  the  numbers. 
As  the  science  of  bacteriology  advances  less  emphasis  is  laid 
upon  quantitative  estimation,  for  the  reason  that  it  is  impossible 
to  gauge  the  quality  of  a  water  only  by  such  estimation.  The 
character  of  the  organisms  present  and  the  relative  abundance  of 
each  species  is  of  more  importance  than  quantitative  estimations. 
Such  estimations  of  water  bacteria,  based  upon  the  counting  of 
colonies  in  plate  cultures,  are  of  little  value,  and  are  in  no  sense 
an  adequate  bacteriological  examination  of  a  water.  It  is  such 
"examinations"  which  have  brought  bacteriology  into  disrepute, 
for  it  is  certain  that  estimations  of  this  kind  are  frequently  not  even 
approximately  correct,  nor  do  they  furnish  any  final  indication  as  to 
safety  or  otherwise  of  a  water  supply.  At  the  same  time  it  should 
not  be  forgotten  that,  other  things  being  equal  and  constant,  a  low 
number  of  organisms  tends  to  indicate  that  a  water  has  not  been 
contaminated  with  organic  matter  or  the  addition  of  foreign  bacteria, 
and  has  not  been  in  a  condition  to  favour  multiplication  of  bacteria, 
and  vice  versd.  Broadly  speaking,  it  must  be  true  that  a  water 
containing  a  large  degree  of  organic  matter,  the  pabulum  of  bacteria, 
will  contain  a  higher  number  of  bacteria  than  a  water  containing  a 


44  BACTERIA  IN  WATER 

low   degree,   and   this,   of   course,   is    the    reason    for    quantitative 
estimations. 

Quality  of  Water  Bacteria 

The  species  of  bacteria  found  in  water  vary  widely.  Many  of 
them  are  common  in  pure  water,  and  may  be  strictly  termed 
"  water  bacteria " ;  others  are  as  clearly  "  sewage  bacteria,"  with 
an  allied  group  belonging  to  the  soil  and  washed  into  rivers,  or 
wells,  by  rain,  and  which  may  be  described  as  "surface  bacteria"; 
and  a  third  group  are  the  pathogenic  bacteria,  which  have  under 
exceptional  conditions  been  isolated  from  water.  Prof.  Marshall 
Ward,  in  his  fifth  report  to  the  Water  Kesearch  Committee  of  the 
Koyal  Society,  drew  up  a  classification  of  water  bacteria,*  which  was 
adopted  two  years  later  by  Boyce  and  HilLf  In  1899  Johnson 
and  Fuller  made  other  groups,!  and  many  other  workers  have  sug- 
gested classifications.  The  two  most  recent  have  been  constructed 
by  Horrocks  of  Net  ley  §  and  Jordan  of  Chicago.  (| 

Both  authorities  recognise  that  provisional  classification  is  all  that 
is  at  present  possible.  Their  groups  are  as  follows : — 


CLASSIFICATION  OF  HORROCKS. 

GROUP 

i.  Fluorescent  bacilli. 

ii.  B.  aquatilis  sulcatus. 

iii.  B.  subtilis  and  "  Potato  bacilli." 

iv.  B.  liquefaciens. 

v.  Chromogenic  (red)  bacilli. 

vi.  Chromogenic  (yellow)  bacilli. 

vii.  Chromogenic  (blue)  bacilli, 

viii.  Chromogenic  (milk-white)  bacilli. 

ix.  Chromogenic  (brown)  bacilli. 

x.  Micrococci. 

xi.  Sarcinae. 

xii.  Spirilla. 

xiii.  Denitrifying  and  nitrifying  bac- 
teria. 

xiv.  B.  coll  communis. 

xv.  B.  enteritidis  sporogenes. 

xvi.  Staphylococci. 

xvii.  Streptococci, 

xviii.  The  Proteus  group. 

xix.  Sewage  bacteria. 

xx.  B.  typhosus. 


CLASSIFICATION  OF  JORDAN. 
GROUP 

i.  B.  coli  communis. 
ii.  B.  lactis  aerogenes. 
iii.  Proteus, 
iv.  B.  enteritidis. 
v.  B.  fl,uorescens  liquefaciens. 
vi.  B.  fluorescens  non-liquefacients. 
vii.  B.  subtilis. 

viii.  Non-gas  forming,  non-fluorescent, 
non-sporulating,    liquefy    gela- 
tine and  acidify  milk, 
ix.  Similar  to  Group  viii.,  but  milk 

rendered  alkaline. 
x.  Similar  to  Group  viii. ,  but  gelatine 

not  liquefied. 
xi.  Similar  to  Group  ix. ,  but  gelatine 

not  liquefied, 
xii.  Similar    to    Group    xi.,   but    the 

reaction  of  milk  not  altered, 
xiii.  Chromogenic  bacilli,  not  included 

in  above  groups. 

xiv.  Chromogenic  Staphylococci. 
xv.  Non-chromogenic  Staphylococci. 
xvi.  Sarcinse. 
xvii.  Streptococci. 


*  Proc.  Roy.  Soc.,  1897,  Ixi.,  p.  415. 

t  Jour,  of  Path,  and  Bact.,  1899,  vi.,  p.  32. 

J  Jour,  of  Exp.  Med.,  1899,  iv.,  p.  609. 

§  An  Introduction  to  the  Bacteriological  Examination  of  Water,  1901,  p.  42  et  sey. 

II  Jour,  of  Hygiene,  1903,  vol.  iii.,  tfo.  1,  p.  5. 


QUALITY  OF  WATER  BACTERIA  45 

Both  the  above  quoted  authorities  furnish  a  large  body  of  facts 
illustrative  of  the  characteristics  of  the  various  groups  suggested,  to 
which  the  reader  is  referred  for  further  particulars.  Broadly  it  may 
be  said  that  the  organisms  classified  in  twenty  groups  by  Horrocks 
are  divisible  into  a  few  general  divisions.  Groups  i.-xii.  are  the 
ordinary  water  bacteria  ;  Group  xiii.  is  the  denitrifying  and  nitrify- 
ing organisms  found  in  soil,  water,  etc.;  Groups  xiv.-xix.  are  the 
sewage  bacteria ;  and  Group  xx.  represents  the  pathogenic  group  of 
organisms  occurring  occasionally  in  water.  Brief  reference  will 
now  be  made  to  these  four  groups,  with  the  exception  of  the  second, 
which  will  be  dealt  with  subsequently. 

(a)  Ordinary  Water  Bacteria. — These  are  organisms  usually 
found  in  pure  or  approximately  pure  waters.     They  are  common  in 
well  waters  and  unpolluted  river  water.     They  include  the  common 
fluorescent  bacilli,  liquefying  and  non-liquefying,  and  which  create 
an  iridescent  green  colour  in  the  nutrient  media.     In  this  class  also 
are    B.    aquatilis    sulcatus,   the   "potato    bacilli"   (B.    mesentericus, 
vulgatus,  fuscus,  et  ruler),  the  "  hay  bacilli "  (B.  subtilis,  B.  mycoides, 
B.  megatherium),  the  liquefying  bacilli  common  in  unfiltered  waters, 
the   chroinogenic   organisms   (B.  prodigiosus,  B.  lactis  erythrogenes, 
B.  rubescens,  B.  arborescens,  B.  aquatilis,  B.  aurantiacus,  B.  violaceus, 
etc.),  and  the  micrococci,  sarcinae,  and  ordinary  water  spirilla.*     The 
presence   of   these   species    of    bacteria    in   water,   unless   in   very 
exceptional   numbers,   indicates   little   of   importance.      They   vary 
according  to  season,  geological  formation  over  or  through  which  the 
water   passes,    surface   washings,   aeration   of   the  water,  forms   of 
vegetation  existing  in  the  water,  and   many  other  similar  natural 
conditions.     The  fluorescent  and  non-gas-producing  and  non-liquefy- 
ing bacilli  are  generally  less  abundant  in  recently  polluted  waters 
than    in    purer   waters,   and   non-chromogenic   staphylococci   more 
abundant. 

(b)  Sewage  Bacteria. — This  group  includes  B.  coli  communis  and 
its  allies,  the  Proteus  family,  B.  enteritidis  sporogenes  of  Klein,  and 
certain   streptococci   and   staphylococci.     They   will    be  treated   of 
subsequently  in  a  chapter  devoted  to  the  bacteriology  of  sewage  (see 
pp.  152-157).     Exception  will,  however,  be  made  in  the  case  of  B.  coli 
communis,  as  this  organism  is  perhaps  the  most  important  in  relation 
to  water.     It  will,  therefore,  be  considered  here.     In  the  first  place 
the  chief  biological  and  cultural  facts  may  be  stated,  and  in  the 
second  place  a  general  note  may  be  added. 

*  The  biological  characters  of  these  various  groups  of  water  bacteria  will  be 
found  in  Frankland's  Micro-organisms  in  Water,  pp.  399-508 ;  Lehmann  and 
Neumann's  Bacteriology,  vol.  ii.,  pp.  133-381;  Crookshank's  Bacteriology  and 
Infective  Diseases  ;  Horrocks'  Bacteriological  Examination  of  Water,  pp.  42-80  ;  and 
in  the  systematic  works  of  Sternberg,  Fliigge,  Besson,  Mace,  etc. 


46  BACTERIA  IN  WATER 


BACILLUS  COLI  COMMUNIS  (Escherich) 

Source  and  habitat — An  organism  of  wide  distribution,  normally  present  in  the 
excreta  of  man  and  animals.  Abundant  in  crude  sewage  (100,000  per  c.c.  in 
London  Sewage,  Houston).  In  polluted  water,  milk,  soil,  etc. 

Morphology — A  short  rod  with  round  ends;  size  and  shape  may  vary  in  same 
colony;  polymorphism,  depending  upon  age  of  culture,  products  of  culture,  com- 
position of  medium,  etc.,  2  to  3  /z  long,  0'5  to  0'6  IM  broad ;  sometimes  oval,  hardly 
longer  than  broad.  Usually  single,  but  occasionally  in  pairs,  bundles,  or  even 
chains  and  threads  (Plate  3). 

Staining  reaction— Ordinary  aniline  dyes.  Decolorised  by  Gram.  (Schmidt 
states  that  B.  coli  from  fatty  stools  of  infants  holds  the  Gram.) 

Capsule — Present. 

Flagella—3  or  4  in  number,  fragile,  short,  and  not  wavy.  Sometimes  only  a 
terminal  one ;  sometimes  several  long  ones  ;  but  polar  staining  and  vacuolation 
frequently  present  in  old  cultures,  or  cultures  grown  under  unfavourable  con- 
ditions. 

Motility — Present,  especially" in  young  cultures,  but  not,  as  a  rule,  so  active  as 
B.  typhosus  ;  oscillatory  rather  than  progressive.  Sometimes  apparently  absent. 

Spore  formation — None. 

Biology :  cultural  characters  (including  biochemical  features} — Grows  best  at  37°  C. , 
but  will  also  grow  at  room  temperature.  Gordon  showed  that  many  varieties  of 
B.  coli  exist  with  many  minor  modifications  (Jour,  of  Path,  and  Bact.,  1897). 

In  gelatine  plate  cultures  the  colonies  appear  generally  within  24  hours  at  20°  C. 
The  deep  colonies  appear  as  small  white  dots,  the  surface  colonies  as  delicate, 
slightly  granular  films  of  an  irregularly  circular  shape.  They  are  bluish-white  by 
reflected,  and  amber  colour  by  transmitted  light.  The  diameter  of  the  colony  is 
1  to  2  mm.  The  colonies  are  transparent,  and  sometimes  iridescent,  especially 
towards  the  periphery,  but  at  the  centre  and  over  the  entire  surface  in  old  cultures 
an  opacity  due  to  a  greater  thickness  of  the  bacterial  growth  is  observed  (Plate  4). 

It  has  been  observed  that  species  derived  from  water  grow  in  transparent 
colonies,  whereas  those  from  the  alimentary  canal  or  excreta  may  show  opacity  of 
the  colony,  which  characteristic  disappears  if  the  culture  be  passed  through  milk. 
About  the  second  or  third  day  the  surface  colonies  attain  a  diameter  of  5  to  6  mm. , 
and  become  marked  by  concentric,  or  radiating,  or  irregular  markings.  The 
surrounding  gelatine  very  frequently  acquires  a  dull,  cloudy,  faded  appearance, 
and  the  edges  of  the  colony  become  more  crenated  and  thinner.  The  whiteness  of 
±he  colony  turns  to  yellow.  There  is  no  liquefaction  of  the  gelatine. 

In  gelatine  stab-cultures  the  organism  grows  rapidly.  On  the  surface,  in  twenty- 
four  hours,  the  growth  is  often  2  to  3  mm.  in  diameter,  and  closely  resembles  a 
surface  colony  in  a  plate  culture,  though  more  luxuriant.  A  thick  white  growth 
extends  along  the  whole  length  of  the  track  of  the  needle,  and  not  infrequently  gas 
bubbles  or  fissures  appear.  The  gelatine  is  not  liquefied,  even  in  old  cultures. 

In  gelatine  streak  cultures  growth  is  also  abundant.  In  twenty-four  hours  the 
elongated  milky  surface  colony  may  be  5  mm.  in  diameter.  It  consists  of  a  delicate 
faintly-granular  film  with  transparent  and  irregular  margins.  Down  the  centre 
longitudinally  the  growth  is  thicker  and  therefore  more  opaque.  Irregular  thick- 
enings, foldings,  and  corrugations  may  occur  in  old  cultures.  Sometimes  the  film 
shows  iridescence,  and  the  medium,  though  not  liquefied,  becomes  clouded.  The 
growth,  as  on  the  plate  cultures,  is  bluish-white  by  reflected,  and  yellowish-amber 
in  colour  by  transmitted  light. 

In  25  per  cent,  gelatine  at  37°  C.— In  48  hours  the  melted  gelatine  remains  clear, 
but  a  thick  pellicle  forms  on  the  surface  (Klein). 

Gelatine  shake  cultures  become  turbid,  and  within  twenty-four  hours  at  20°  C.  are 
riddled  with  bubbles  of  gas,  which  are  generally  more  numerous  and  larger  towards 
the  foot  of  the  tube.  They  increase  in  size  by  the  second  day,  sometimes  even 
forming  fissures.  The  gas  is  mainly  carbonic  acid.  The  presence  of  a  small  per 
cent,  of  fermentable  sugar  in  the  medium  increases  the  gas  production  (Plate  4.). 
On  potato-gelatine  the  colonies  of  B.  coli  are  similar  in  appearance  to  those 


PLATE  3. 


Bacillus  coli  communis.    From  agar  culture,  48  hours  at  37°  C. 


x  1000. 


Proteus  vulgaris.    Impression  preparation  from  "  swarming  islands"  on  gelatine, 
20  hours  at  20°  C.     X   3000. 


[To  face  page 


BACILLUS  COLI  47 

occurring  on  ordinary  gelatine,  except  that  they  grow  more  slowly,  are  more 
circumscribed,  and  of  a  characteristic  brown  colour  (Houston). 

On  carbol-gelatine  ('05  per  cent,  of  phenol),  the  growth  does  not  differ  from 
ordinary  gelatine  cultures  except  that  it  is  delayed. 

Broth— In  less  than  twelve  hours  at  37°  C.  the  medium  becomes  uniformly 
turbid.  It  may  be  very  pronounced.  Frequently  there  is  also  at  a  later 
stage  a  marked  amorphous  flocculent  sediment  consisting  of  bacteria.  Only  a 
faint  film  forms  on  the  surface,  which  rarely  becomes  a  pellicle.  There  is  a  foetid 
odour,  and  sometimes  gas  formation.  In  glucose,  lactose,  saccharose  broth 
(2  per  cent),  and  glucose-formate  broth  (Pakes),  and  bile-salt  broth  (M'Conkey), 
the  growth  is  abundant,  and  gas  is  produced.  In  phenolated  broth  ('05  per 
cent,  of  phenol),  and  in  broth  containing  formalin  (1  to  7000),  there  is 
also  growth. 

On  agar  at  37°  C.  the  organism  grows  rapidly,  producing  thin,  moist,  translucent 
creamy  greyish-white  colonies  of  irregular  shape  and  size.  The  colonies  grow  more 
rapidly  on  the  surface  than  in  the  depth  of  the  medium.  The  same  appearances 
occur  on  agar  at  20°  C. ,  except  that  the  growth  is  delayed.  Gas  bubbles  frequently 
occur  in  the  condensation  fluid. 

Litmus  lactose  agar  (2  per  cent.) — The  medium  is  turned  red  in  twenty-four  hours, 

B.  Typlwsus.    .  B.  coli. 


FIG.  9.— Diagrams  of  B.  typhosus  and  B.  coli. 

and  the  surface  growth  becomes  tinged  slightly  with  the  reddened  litmus.     Numer- 
ous gas  bubbles  are  produced  in  the  medium. 

On  potato  at  37°  C.  there  is  produced  in  twenty-four  hours  a  thick,  moist, 
yellowish-grey  growth,  becoming  brown  in  old  cultures.  The  colour  varies  widely 
in  degree,  sometimes  being  richer  than  at  other  times.  The  potato  becomes  changed 
in  colour  near  the  growth.  If  the  potato  is  not  fresh,  or  its  reaction  has  been  made 
alkaline,  the  growth  of  B.  coli  may  be  almost  colourless.  There  are,  of  course,  a 
very  large  number  of  bacteria  which  produce  a  growth  on  potato  not  readily 
distinguishable  from  B.  coli. 

Litmus  milk — Usually  an  acid  curdling  of  the  milk  occurs  in  twenty-four  hours  at 
37°  C.,  though  sometimes  slightly  delayed.  The  bluish-purple  colour  changes  to 
pink,  then  the  whole  of  the  milk  is  turned  into  a  solid  compact  coagulum,  the  milk 
itself  becoming  white.  Later  the  redness  extends  from  the  surface  downwards 
until  the  whole  contents  of  the  tube  are  bright  red  in  colour. 

On  blood  serum  at  37°  C.  an  abundant  white  glistening  layer  is  rapidly  developed, 
somewhat  similar  to  the  growth  on  agar.  There  is  no  liquefaction. 

water).      The  reaction 

-eight  hours  at  37°  C.,  but  in  any  case  is  generally 
kept  at  37°  C.  for  five  days.  The  "red  reaction  " 
may  be  obtained  by  adding  to  such  a  culture  1  c.c.  of  a  0'02  per  cent  solution  of 
potassium  nitrite,  and  0'5  c.c.  of  strong  sulphuric  acid.  If  the  colour  (due  to 


48 


BACTERIA  IN  WATER 


nitroso-indol)  does  not  appear  at  once,  the  culture  may  be  incubated  for  a  brief 
period. 

Reduction  of  nitrate. — B.  coli  is  a  vigorous  denitrifying  organism.  In  twenty- 
four  hours  at  37°  C.  the  reduction  of  nitrates  to  nitrites  is  well  marked.  (Bouillon 
5  per  cent.,  KNO3  0*1  per  cent.,  water  94*9  per  cent.). 

Aerobic  or  facultative  anaerobic. 

Vitality  and  powers  of  resistance,  not  considerable,  but  more  than  the  typhoid 
bacillus. 

The  following  table  of  comparative  features  of  B.  coli  and  B.  typhoons  is  a 
provisional  scheme  of  some  of  the  differences  between  a  typical  B.  coli  and  a 
typical  typhoid  bacillus.  As  is  pointed  out  elsewhere,  the  Coli  group  is  large  and 
its  characteristics  vary  according  to  origin,  race,  cultivation,  and  many  other 
conditions.  In  some  ways  the  table  is  misleading,  as  it  is  exceptional  to  find  a 
bacillus  which  gives  all  these  features,  but  the  table  is  inserted  for  reference, 
because  in  a  general  way  it  states  the  broad  differences  between  the  types  : — 


Comparative  Features 

B.  typhosus. 
Morphology— Bacillus  of  unequal  lengths; 

some  filaments. 
Flagella — Long,  wavy,  spiral,  numerous 

(9  to  18) ;  movement  very  active. 

On  gelatine  and  agar — Angular,  irregular, 

slightly  raised  colonies  ;  slow  growth  ; 

medium  remains  clear. 
In  gelatine — In  ordinary  gelatine  and  in 

lactose  gelatine  no  gas  is  produced  (at 

20°  CA     No  liquefaction. 
Milk — Not  curdled  by  the  bacillus  (at 

37°  C.).     No  acid  production. 
Indol — In  bouillon  and  Witte's  peptone 

water,  no  production  of  indol. 
Bouillon  containing  0'3  per  cent.  Phenol, 

or  Formalin  (1  :  7000) — No  growth. 
Lactose — bouillon  at  37°  C. — No  gas  pro- 
duction. 

Neutral-red  glucose-agar — No  change. 
Glucose  or  lactose  media,  shake  cultures — 

No  gas  production. 
Potato— An  "invisible  growth"  if  the 

potato  is  acid  in  reaction. 
25  per  cent,  gelatine  at  37°  0.—  Strongly 

and  uniformly  turbid    (Klein).       No 

pellicle. 
Eisner's  iodised    potato-gelatine  —  Slow 

growth  ;  small  transparent  colonies. 
Proskauer  and  Capaldis  Medium,  No.  1 

— No  growth  ;  no  change  in  reaction. 
Widal's  reaction— Bacilli  became  motion- 
less and  agglutinated  when  suspended 

in  blood  serum  from  a  typhoid  patient. 

(See  Appendix.) 
M'Conkey's lactose  agar — Surface  colonies 

transparent ;  medium  clear. 
Vitality  in  icater  or  sewage— B.  typhosus 

soon  ceases  to  multiply  and  more  or 

less  readily  dies. 
Pfeiffer's  inoculation  test  with  anti-typhoid 

serum — Negative  result. 


of  B.  coli  and  B.  typhosus 

B.  coli. 

Bacillus  shorter  and  thicker;  filaments 
rare. 

Shorter,  stiffer,  few  (average  3),  move- 
ment less  active,  and  sometimes  almost 
absent. 

Colonies  with  even  margin,  homogenous, 
much  larger  and  quicker  growth, 
medium  becomes  turbid  or  coloured. 

Under  the  same  circumstances  abundant 
gas  is  produced.  No  liquefaction. 

Milk  is  curdled,  within  24  to  48  hours  at 

37°  C.     Abundant  acid  production. 
Indol  is  present  as  a  rule. 

Grows  well  and  uniformly  throughout 

medium. 
Gas  production  occurs. 

Marked  green  fluorescence. 
Marked  gas  production. 

Thick,  yellowish-white  growth,  later  be- 
coming brown  in  colour. 

Gelatine  remains  clear  within  48  hours, 
but  a  thick  pellicle  forms  on  the 
surface. 

Rapid  growth  ;  large  brown  colonies. 

Growth  ;  acid  reaction. 

B.  coli  remains  motile  and  not  aggluti- 
nated. 


Surface    colonies    white    with    yellow 

centre ;  haze  on  medium. 
B.   coli  retains    vitality  and   power  of 

self-multiplication . 

Positive  result,  variable  symptoms  ac- 
cording to  virulence  of  bacillus. 


ARTIFICIAL  PURIFICATION  OF  WATER  65 

bicarbonate  of  lime,  is  converted  into  insoluble  normal  carbonate  of 
lime  by  the  addition  of  a  suitable  quantity  of  limewater.  Carbonates 
of  lime  and  magnesia  are  soluble  in  water  containing  free  carbonic 
acid,  but  when  fresh  lime  is  added  to  such  water  it  combines  with  the 
free  C02  to  form  the  insoluble  carbonate,  which  falls  as  a  sediment : — 

CaCO3  +  CO2  +  CaH2O2  (limewater)  =  2  CaCO3  +  H2O. 

As  the  carbonate  falls  to  the  bottom  of  the  tank  it  carries  down 
with  it  the  organic  particles.  Hence  sedimentation  is  brought  about 
by  means  of  chemical  precipitation.  It  is  obviously  a  mechanical 
process  as  regards  its  action  upon  bacteria.  Nevertheless  its  action 
is  well-nigh  perfect,  and  400  bacteria  per  c.c.  may  be  reduced  to  4 
or  5  per  c.c.  We  shall  refer  to  this  same  action  when  we  come  to 
speak  of  bacterial  purification  of  sewage.  Alum  has  been  frequently 
used  to  purify  water  which  contain  much  suspended  matter.  Five 
or  six  grains  of  alum  are  added  to  each  gallon  of  water,  plus  some 
calcium  carbonate  by  preference.  Precipitation  occurs,  and  with  it 
sedimentation  of  the  bacteria,  as  before.  But,  as  Babes  has  pointed 
out,  alum  itself  acts  inimically  on  germs ;  in  such  treatment,  there- 
fore, we  get  sedimentation  and  germicidal  action  combined. 

As  a  matter  of  actual  practice,  however,  sedimentation  alone  is 
rarely  sufficient  to  purify  water.  It  is  true  that  the  collection  of 
water  in  large  reservoirs  permits  subsidence  of  suspended  matters, 
affords  time  for  the  action  of  light,  and  the  suicidal  competition 
among  the  common  water  bacteria.  But  in  small  collections  of 
water  it  is  otherwise.  Here  filtration  is  the  most  important  and 
most  reliable  method. 

Sand  filtration,  as  a  means  of  purifying  water,  has  been  practised 
since  the  early  part  of  last  century.  But  it  was  not  till  1885  that 
Percy  Frankland  first  demonstrated  the  great  difference  in  bacterial 
content  between  a  water  unfiltered  and  a  water  which  had  passed 
through  a  sand  filter  (only  about  3  per  cent,  of  the  bacteria  originally 
present  being  left  in  the  water).  Previous  to  this  time  the  criterion 
of  efficiency  in  water  purification  had  been  a  chemical  one  only, 
and  the  presence  or  absence  of  bacteria  in  any  appreciable  quantity 
was  described  not  in  mathematical  terms,  but  in  indefinite  descriptive 
words,  such  as  "  turbid,"  "  cloudy,"  etc.  It  is  needless  to  say  that  this 
difference  in  estimation  was  largely  due  to  the  introduction  by  Koch 
of  the  gelatine-plate  method  of  examination.  As  a  result  of  investi- 
gation Percy  Frankland  formulated  the  following  conclusions  as 
regards  the  chief  factors  influencing  the  number  of  microbes  passing 
through  the  filter.  The  efficiency  of  filtration,  he  held,  depended 
upon  (a)  the  storage  capacity  for  unfiltered  water,  by  which  it  was 
possible  to  obtain  the  preliminary  advantage  of  subsidence ;  (b)  the 
thickness  of  fine  sand  through  which  the  filtration  is  carried  on; 

E 


66  BACTERIA  IN  WATER 

(e)  rate  of  filtration ;  (d)  the  renewal  of  the  filter-beds.  After  a 
certain  time  the  filter-bed  becomes  worn  out  and  inefficient,  and  at 
such  times  renewal  is  necessary.  Not  only  may  the  age  of  the 
filter  act  prejudicially,  but  the  extra  pressure  required  will  tend  to 
force  through  it  bacteria  which  ought  to  have  remained  in  the  filter. 

In  1890  a  special  study  of  filtration  was  made  by  the  Massa- 
chusetts State  Board  of  Health,  and  in  annual  reports  published 
from  1890,  a  number  of  experiments  are  recorded  which  have  proved 
of  classic  importance,  and  which  should  be  consulted  by  the  student 
or  practical  worker  desiring  to  acquire  a  thorough  grasp  of  the 
principles  of  biological  filtration.  There  it  is  shown  that  water  can 
be  filtered  through  sand  filters  at  the  rate  of  3,000,000  gallons  per 
acre  daily  and  9 9 '9 5  per  cent,  of  the  bacteria  removed.  In  actual 
practice  it  was  found  that  the  finer  sands  were  more  effective  than 
the  coarser,  and  under  moderate  pressure  1  foot  of  sand  was  as  effec- 
tive as  5  feet.  Over  80  per  cent,  of  the  bacteria  removed  were 
found  in  the  upper  inch  of  sand  and  55  per  cent,  in  the  upper 
quarter  inch.  If  the  surface  of  the  filter  was  scraped,  it  was  shown 
that  an  increased  number  of  bacteria  passed  through  the  filter,  which 
was  therefore  much  less  effectual.  Subsequently,  Koch  emphasised 
the  importance  of  this  vital  layer.  But  it  was  the  Massachusetts 
Board  that  first  proved  by  experiment  that  the  oxidation  which  occurs 
in  a  filter-bed  was  due  to  the  nitrifying  organisms  in  the  surface 
or  scum  layer.  When  nitrification  is  established  in  a  filter,  the  rate 
of  filtration  within  certain  limits  was  found  to  exert  comparatively 
little  effect  upon  the  removal  of  the  organic  matter. 

In  1893  Koch  brought  out  his  monograph  upon  Water  Filtration 
and  Cholera,  and  his  work  had  a  deservedly  great  influence  upon  the 
whole  question.  He  showed  how  the  careful  filtration  of  water 
supplied  to  Altona  from  the  Elbe  saved  the  town  from  the  epidemic 
of  cholera  which  came  upon  Hamburg  as  a  result  of  drinking 
unfiltered  water,  although  Altona  is  situated  several  miles  below 
Hamburg,  and  its  drinking  water  is  taken  from  the  river  after  it  has 
received  the  sewage  of  the  latter. 

Now,  from  his  experience  of  water  filtration,  Koch  arrived  at 
several  important  conclusions.  In  the  first  place,  he  maintained 
that  the  portion  of  the  filter-led  which  really  removed  micro-organisms 
effectively  was  the  slimy  membranous  organic  layer  upon  the  surface  of 
the  sand.  This  layer  is  produced  by  a  deposit  from  the  still  unpurified 
water  lying  immediately  above  it.  The  most  vital  part  of  the  filter- 
bed  is  this  organic  layer,  which,  after  formation,  should  not  be  dis- 
turbed until  it  requires  removal  owing  to  its  impermeability.  A 
filter-bsd,  as  is  well-known,  consists  of,  say,  3  feet  of  sand  and  1 
foot  of  coarse  gravel.  The  water  to  be  filtered  is  collected  into  large 
reservoirs,  where  subsidence  by  gravitation  occurs.  From  thence  it 


FILTRATION  OF  WATER  67 

is  led  by  suitable  channels  to  the  surface  of  the  filter-bed.  Having 
passed  through  the  3  or  4  feet  of  the  bed,  it  is  collected  in  a  storage 
reservoir  and  awaits  distribution.  Such  being  the  principles  of 
construction,  it  will  be  apparent  that  the  action  of  the  whole  process 
is  both  mechanical  and  chemical.  Mechanically  by  subsidence, 
much  suspended  matter  is  left  behind  in  the  reservoir.  Again, 
mechanically,  much  of  that  which  remained  suspended  in  the  water 
when  it  reached  the  filter-bed  is  waylaid  in  the  substance  of  the 
sand  and  gravel  of  the  filter-bed.  The  next  change  is  a  chemical 
one.  Oxidation  of  the  organic  matter  occurs  to  some  extent  as  the 
water  passes  through  the  sand.  Until  recently  this  chemical  action 
and  the  double  mechanical  action  (sedimentation  and  straining)  was 
believed  to  be  the  complete  process,  and  its  efficiency  was  tested  by 
chemical  oxidation  and  alteration,  and  absence  of  the  suspended 
matter.  Now,  however,  it  is  recognised  that  the  second  portion  of 
the  chemical  action  is  vastly  the  more  important,  indeed,  the  only 
vital  part  of  the  process.  This  is  the  chemical  effect  of  the  layer 
of  scum  and  mud  on  the  surface  of  the  sand  at  the  top  of  the  filter- 
bed.  The  mechanical  part  of  this  layer  is,  of  course,  the  holding 
back  of  the  particulate  matter  which  has  not  subsided  in  the  reservoir ; 
the  vital  action  consists  in  what  is  termed  nitrification  of  unoxidised 
substance,  which  is  accomplished  in  this  layer  of  organic  matter. 
We  shall  deal  at  some  length  with  the  principles  of  nitrification 
when  we  come  to  speak  of  soil.  But  we  may  say  here  that  by 
nitrification  is  understood  a  process  of  oxidation  of  elementary 
compounds  of  nitrogen,  by  which  these  latter  are  built  up  into  stable 
bodies  which  can  do  little  or  no  harm  in  drinking-water.  The  action 
of  a  filter-bed  may,  therefore,  be  summarised  as  follows : — There  is 
(1)  subsidence  of  the  grosser  particles  of  impurity  in  the  settling 
tank ;  (2)  mechanical  obstruction  to  impurities  in  the  interstices  of 
the  scum,  sand,  and  gravel  in  the  filter;  (3)  oxidation  of  organic 
matter  by  the  oxygen  held  in  the  pores  of  the  sand  and  gravel; 
(4)  nitrification  in  the  vital  scum  layer,  which  is  accomplished  by 
micro-organisms  themselves.  This  latter  is  now  considered  to  be 
incomparably  the  most  important  part  of  the  filter.  That  being  so, 
its  removal,  except  when  absolutely  necessary,  is  to  be  avoided  as 
detrimental  to  the  efficiency  of  the  filter.  New  filters  have  obviously 
but  little  of  this  action.  Kiimmel  found  that  when  a  filter  had  new 
sand  placed  upon  it  the  number  of  bacteria  in  the  filtered  water  was 
as  follows:-  Perc>c- 

Before  cleaning     .  .  42 


One  day  after  cleaning    . 
Two  days  after  cleaning  . 
Three  days  after  cleaning 
Four  days  after  cleaning 
Five  days  after  cleaning 
Six  days  after  cleaning     . 


1880 
752 
208 
156 
102 
84 


68  BACTERIA  IN  WATER 

Hence  it  is  necessary  to  allow  a  new  filter-bed  to  act  for  a  short 
period  (say  four  days)  before  the  filtered  water  is  used  for  domestic 
purposes,  in  order  to  allow  a  fresh  film,  the  organic  layer,  to  be  formed. 
This  must  also  be  borne  in  mind  after  a  filter-bed  has  been  cleaned.* 

To  maintain  this  nitrifying  action  of  a  filter  in  efficiency,  Koch 
suggested,  in  the  second  place,  that  the  rate  of  filtration  must  not 
exceed  four  inches  per  hour.  At  the  Altona  water-works  this  rate  of 
filtration  was  maintained,  and  the  number  of  organisms  always 
remained  below  100  per  c.c.,  which,  as  we  have  seen,  is  the  standard. 
Thirdly,  it  is  important  that  periodic  bacteriological  examinations 
should  be  made.  Koch's  emphasis  upon  this  point  is  well  known,  and 
the  cumulative  experience  of  bacteriologists  only  further  supports  such 
a  course  being  taken.  Clark  and  Gage  of  the  Lawrence  Experimental 
Station,  claim  that  the  test  for  the  presence  of  B.  coli  is  a  more  delicate 
indication  of  filter  efficiency  when  filtering  polluted  water  than  tests  for 
the  total  number  of  bacteria  present.  Fourthly,  Koch  maintained  that 
the  thickness  of  the  sand  of  the  filter-bed  should  never  be  less  than  one 
foot.  Fifthly,  if  it  be  true  that  efficient  sand  filtration  is  a  safeguard 
against  putrefactive  and  disease-producing  germs,  then  there  can  be 
but  one  criterion  of  efficiency,  viz.,  their  absence  in  the  filtered  water, 
which  can  only  be  ascertained  by  regular  examination.  But  it  is 
not  alone  for  pathogenic  germs  that  filtration  is  proposed.  Hence 
Koch  laid  down  that  filtered  water  containing  more  than  100  micro- 
organisms of  any  kind  per  c.c.  is  below  the  standard  of  purity,  and 
should  not,  if  possible,  be  distributed  for  drinking  purposes.  In  this 
country  chemical  analysis,  with  a  more  or  less  cursory  microscopic 
examination,  has  been  almost  invariably  accepted  as  reliable  indication 
of  the  condition  of  the  water.  But  such  an  examination  is  not  really 
any  more  a  fair  test  of  the  working  of  the  filter  than  it  is  of  the 
actual  condition  of  the  water.  It  is  true,  the  quantity  of  organic 
matter  can  be  estimated  and  the  condition  in  which  it  exists  in 
combination  obtained;  but  it  cannot  tell  us  what  a  bacteriological 
examination  can  tell  us,  viz.,  the  quantity  and  quality  of  living 
micro-organisms  present  in  the  water.  Upon  this  fact,  after  all,  an 
accurate  conclusion  depends.  There  is  abundant  evidence  to  show 
that  no  valuable  opinion  can  be  passed  upon  a  water  except  by  both 
a  chemical  and  a  bacteriological  examination,  and  further  by  a 
personal  investigation,  outside  the  laboratory,  of  the  origin  of  the 
water  and  its  liabilities  to  pollution. 

So  convinced  was  Koch  of  the  efficiency  of  sand  filtration  as 
protection  against  disease-producing  germs,  that  he  advocated  an 
adaptation  of  this  plan  in  cases  where  it  was  found  that  a  well 
yielded  infected  water.  Such  pollution  in  a  well  may  be  due  to 

*  See  also  Thirty-fourth  Ann.  Rvp.  State  Ed.  of  Health,  Massachusetts,  1903, 
p.  228. 


FILTRATION  OF  WATER 


69 


various  causes ;  surface-polluted  water  oozing  into  the  well  is  probably 
the  commonest,  but  decaying  animal  or  vegetable  matter  might  also 
raise  the  number  of  micro-organisms  present  almost  indefinitely. 
Koch's  proposal  for  such  a  polluted  well  was  to  fill  it  up  to  its 
highest  water  level  with  gravel,  and  above  that,  up  to  the  surface 
of  the  ground,  with  fine  sand.  Before  the  well  is  filled  up  in  this 
manner  it  must,  of  course,  be  fitted  with  a  pipe  passing  to  the 
bottom  and  connected  with  a  pump.  This  simple  procedure  of  filling 
up  a  well  with  gravel  and  sand  interposes  an  effectual  filter-bed 
between  the  subsoil-water  and  any  foul  surface-water  percolating 
downwards.  Such  an  arrangement  yields  as  good,  if  not  better, 
results  than  an  ordinary  filter-bed,  on  account  of  there  being 
practically  no  disturbance  of  the  bed  nor  injury  done  to  it  by  frost. 
The  evidence  that  filter-beds  remove  pathogenic  bacteria  has  not  only 
been  demonstrated  by  experiment  but  by  actual  experience.  At 
Lawrence,  Hamburg,  Mount  Vernon,  and  other  towns,  a  marked 
decline  in  water-borne  typhoid  fever  has  occurred  as  a  result  of 
filtration. 

The  effect  of  filtration  upon  the  number  of  bacteria  was  demon- 
strated in  the  results  which  Sir  Edward  Frankland  arrived  at  in  his 
investigation  of  London  waters  so  long  ago  as  1887.* 


Mean  of  Monthly  Examinations  for  the    Year. 


Micro-organisms  per  c.c. 

Average  %  of 

Name  of  Company. 

Source  of 
Supply. 

Micro- 
organisms 
removed  by 

At 

After 

After 

Source. 

Storage. 

Filtration. 

Filtration. 

The  Chelsea  Co. 

/Thamesan 
\Hampton  / 

16,138 

1,067 

34 

98-96 

West  Middlesex  Co.  . 

16,138 

1,788 

58 

99-40 

Southwark  &  Vauxhall  Co. 

11 

16,138 

-       80 

97-72 

f  623  ] 

Grand  Junction  Co.  . 

M 

16,138 

2,500 

100  \ 

98-46 

\     96  J 

Lambeth  Co. 

»> 

16,138 

7,820 

75' 

99-50 

In  1899  the  Massachusetts  Board  of  Health  found  that  by 
continuous  filtration  through  45  inches  of  sand  (size  0'23  mm.) 
99*49  per  cent,  of  the  bacteria  were  removed ;  and  by  intermittent 
filtration  99*08  per  cent,  of  the  organisms  were  removed.  In  1902 
the  intermittent  filter  removed  98'7  per  cent,  of  the  total  bacteria, 
99'9  per  cent.  B.  coli,  and  100  per  cent.  B.  typhosus.  The  continuous 

*  Report  on  the  Metropolitan  Water  Supply,  1887. 


70  BACTERIA  IN  WATER 

filter  removed  98'7  per  cent,  of  the  total  bacteria,  99*8  per  cent,  of 
B.  coli,  and  99'9  of  the  typhoid  bacillus.* 

The  teaching  of  these  figures  could,  with  great  ease,  be  emphasised 
again  and  again  if  such  was  necessary ;  but  sufficient  has  been  said 
to  show  that  sand  filtration,  when  carefully  carried  out,  offers  a  more 
or  less  absolute  barrier  to  the  passage  of  bacteria,  whether  non- 
pathogenic  or  pathogenic. 

Domestic  Purification  of  Water 

Something  may,  however,  be  added,  from  a  bacteriological  point 
of  view,  relative  to  what  is  called  domestic  purification.  There  is  but- 
one  perfectly  reliable  method  of  sterilising  water  for  household  use, 
viz.,  boiling.  As  we  have  seen,  moist  heat  at  the  boiling-point  main- 
tained for  a  few  minutes  will  kill  all  bacteria  and  their  spores.  The 
only  disadvantages  to  this  process  are  the  labour  entailed  and  the 
"flat"  taste  of  the  water.  Nevertheless,  in  epidemics  due  to  bad 
water,  it  is  desirable  to  revert  to  this  simple  and  effectual  purification. 

There  are  a  large  number  of  domestic  filters  on  the  market  with, 
in  many  cases,  but  little  difference  between  them.  The  materials 
out  of  which  they  are  made  are  chiefly  the  following:  carbon  and 
charcoal,  iron  (spongy  iron  or  magnetic  oxide),  asbestos,  porcelain 
and  other  clays,  natural  porous  stone,  and  compressed  siliceous  and 
diatomaceous  earths.  From  an  extended  research  in  1894  by  Prof. 
Sims  Woodhead  and  Dr  Cartwright  Wood,  who  repeated  and 
extended  experiments  by  Freudenreich,  Schofer,  and  others,  our 
knowledge  of  the  quality  of  these  substances  as  protectives  against 
bacteria  has  been  largely  increased.-)-  They  concluded  that  a^  filter 
failed  to  act  in  one  of  two  ways.  It  was  either  pervious  to  micro- 
organisms, or  its  power  of  filtering  became  modified  owing  to  (a) 
structural  alteration  of  its  composition,  or  (b)  to  the  growing  through 
of  the  micro-organisms,  which  had  been  demonstrated  by  previous 
workers.  The  conditions  which  chiefly  influence  the  growth  of 
bacteria  through  a  filter  appear  to  be  the  temperature,  the  inter- 
mittent use  of  the  filter,  and  the  species  of  bacteria.  The  higher  the 
temperature  and  the  longer  the  organisms  are  retained  in  the  filter 
the  more  likely  is  it  that  they  will  grow  through,  and  in  the  next 
usage  of  the  filter  appear  in  the  filtrate.  As  to  the  species,  those 
multiplying  rapidly  and  possessing  the  power  of  free  motility  will 
naturally  appear  earlier  in  a  filtrate  than  others.  Woodhead  and 
Wood  concluded  that  out  of  18  different  kinds  of  domestic  filter,  each 
of  which  had  its  supporter,  the  Pasteur-Chamberland  candle  filters 

*  Thirty-fourth  Ann.  Rep.  State  Bd.  of  Health,  Massachusetts,  1903,  pp.  224 
and  269. 

t  Brit.  Med.  Jour.,  1894,  i.,  pp.  1053,  1118,  1182,  1375,  1486. 


DOMESTIC  FILTRATION  OF  WATER 


(composed  of  porcelain  formed  by  a  mixture  of  kaolin  and  other 
clays)  were  the  only  filters  out  of  the  substances  named  above  which 
were  reliable  and  protective  against  bacteria.  They  tested  over  three 
dozen  of  the  Pasteur  filters,  and  "  in  every  case  these  gave  a  sterile 
filtrate."  Pare  cholera  bacillus  in  suspension  (5000  bacilli  to  every 
c.c.)  and  typhoid  bacillus  in  suspension  (8000  per  c.c.)  were  passed 
through  these  filters,  and  not  a  single  bacillus  was  detectable  in  the 
filtrate.  The  Berkefeld  filter  (siliceous  earth)  came  second  on  the 
list  as  an  effective  filter,  and  had  but  the  one  fault  of  not  being  a 
"continuous"  steriliser.  A  certain  Parisian  filter  ("Porcelaine 
d'Amiante "),  made  of  unglazed  porcelain,  ren- 
dered water  absolutely  free  from  bacteria.  Its 
action  was,  however,  very  slow.  Setting  aside 
these  three  efficient  filters,  we  are  face  to  face 
with  the  fact  that  most  filters  do  not  produce 
germ-free  filtrates,  even  though  they  are  nomin- 
ally guaranteed  to  do  so.  It  is  professed  for 
animal  charcoal,  which  is  widely  used,  that  it 
absorbs  oxygen,  and  so  fully  oxidises  whatever 
passes  through  it.  This  may  be  so  at  first,  but 
after  a  little  use  it  does  more  harm  than  good. 
It  appears  to  add  nitrogen  and  phosphates  to 
water,  which  are  both  nutritive  substances  on 
which  bacteria  grow,  and  it  readily  absorbs  im- 
purities from  the  air.  As  a  matter  of  experiment 
and  practice,  it  has  been  found  by  Frankland, 
Woodhead,  and  others,  that  charcoal  actually 
adds  to  the  number  of  germs  after  it  has  been 
in  use  for  some  time. 

Subsequent  experiments  were  made  in  this 
country  by  Lunt  and  Horrocks.  Lunt  working 
in  1897  investigated  the  power  of  the  Berkefeld 
filter  to  intercept  pathogenic  bacteria,  especially 
the  typhoid  bacillus.  He  concluded  as  a  result 
of  his  inquiry  that  on  the  first  day  an  efficiently  sterilised  Berkefeld 
filter  gave  an  absolutely  sterile  filtrate,  but  that  on  the  second  or 
third  day  of  using  some  water  bacteria  passed  through.  For  thirty- 
nine  days  the  B.  coli  did  not  pass  through,  though  the  organism  could 
be  detected  on  the  outside  of  the  filtering  candle.  Lunt  found  that 
the  action  of  the  filter  depended  very  much  upon  its  method  of 
use:  forcing  or  intermittently  pumping  water  through  the  filter 
resulted  in  a  filtrate  containing  bacteria,  whilst  if  the  same  filter  was 
steadily  used  a  germ-free  filtrate  was  obtained.  In  short,  the  result 
of  Limb's  work  was  to  show  the  necessity  for  a  frequent  sterilisa- 
tion of  the  filter,  for  though  it  allows  ordinary  water  bacteria  to 


FIG.  10.— PASTEUR- 
CHAM  BERLAND  FILTER. 

Attached  to  Water 
Supply. 


72  BACTERIA  IN  WATER 

pass  on  the  second  or  third  day,  B.  coli  and  the  typhoid  bacillus 
do  not  appear  with  them  in  the  filtrate  until  a  subsequent  date. 
Probably,  if  reliance  is  to  be  placed  upon  such  a  filter  from  a 
bacteriological  point  of  view,  daily  sterilisation  is  advisable. 

Experiments  of  a  similar  nature  have  been  done  by  Horrocks,* 
who  arrives  at  the  following  conclusions.  First,  the  B.  typhosus  is  not 
able  to  grow  through  the  walls  of  a  Pasteur-Chamberland  candle,  and 
if  proper  care  be  taken  to  prevent  the  direct  passage  of  organisms 
through  flaws  in  the  material  and  imperfections  in  the  fittings,  the 
Pasteur-Chamberland  filter  ought  to  give  complete  protection  from 
water-borne  disease.  Secondly,  typhoid  bacilli  can  grow  through  the 
walls  of  Berkefeld  candles,  the  time  required  for  the  passage  being 
largely  dependent  on  the  nutriment  supplied  to  the  organisms  by  the 
filtering  fluid.  Possibly  the  weakness  of  the  candle  from  a  bacteri- 
ological point  of  view  is  due  to  the  large  size  of  the  lacunar  spaces, 
which  cannot  be  avoided  if  a  fair  delivery  is  to  be  obtained,  but 
which  "  appears  to  militate  against  the  immobilising  and  devitalising 
influences  which  operate  so  strongly  in  filters  made  with  very  narrow 
lacunar  spaces."  Thirdly,  Horrocks  concluded  that  when  a  highly 
polluted  liquid  containing  typhoid  bacilli  is  filtered  through  a 
Berkefeld  candle  the  bacilli  may  appear  in  the  filtrate  in  four  days. 
Consequently,  it  is  necessary  to  sterilise  these  candles  every  third  day. 
The  method  of  sterilisation  of  filters  is  not  washing  or  brushing  or 
any  other  kind  of  cleansing  or  soaking  in  water,  but  by  exposing 
them  to  steam  or  boiling  them. 

*  Bacteriological  Examination  of  Water,  1901,  pp.  273-280. 


CHAPTEE  III 


BACTERIA   IN   THE  AIR 

Methods  of  Examination  of  Air — Conditions  of  Bacterial  Contamination  of  Air : 
(1)  Dust  and  Air  Pollution ;  (2)  Moisture  or  Dampness  of  Surfaces  :  Bacteria 
in  Sewer  Air ;  (3)  the  Influence  of  Gravity ;  (4)  Air  Currents.  The  Relation  of 
Bacteria  to  CO2  in  the  Atmosphere :  in  Workshops,  in  Bakehouses,  in  Rail- 
way Tubes,  in  the  House  of  Commons. 

THE  basis  of  the  usual  methods  in  practice  for  bacterially  examining 
air  is  to  pass  the  air  over  or  through  some  nutrient  medium.  By 
this  means  the  contained  organisms  are  waylaid,  and  finding  them- 
selves under  favourable  conditions  of  pabulum,  temperature,  and 
moisture,  commence  active  growth,  and  thus  reveal  themselves  in 
characteristic  colonies.  These  are  examined  by  the  microscope  and 
sub-culture.  Eeturns  of  the  number  of  bacteria  in  the  sample  taken 
may  be  made  for  the  sake  of  information,  but  little  or  no  conclusion 
of  value  can  be  drawn  from  such  data.  The  standard  recognised  in 
Europe  is  the  cubic  metre  or  litre,  and  one  may  report,  for  example, 
of  the  air  of  a  room  containing  500  or  'more  germs  per  cubic  metre. 

Methods  of  Examination  of  Air 

1.  The  Plate  Method. — Koch  adopted  the  simplest  of  all  the  culture  methods, 
viz^  exposing  a  plate  of  gelatine  or  agar  for  a  longer  or  shorter  time  to  the  air  of 
which  examination  is  desired.  By  gravity  the  suspended  bacteria  fall  on  the  plate 
and  start  growth.  As  a  matter  of  quantitative  exactitude,  this  method  is  not  to  be 
recommended,  but  it  frequently  proves  an  excellent  method  for  qualitative 
estimation.  It  will  be  found  in  practice  that  nutrient  agar  is  better  for  the  purpose 
than  nutrient  gelatine.  Greater  latitude  is  obtained  both  in  point  of  temperature 
and  length  of  incubation,  and  the  result  is  uncomplicated  by  the,  at  times,  very 
rapid  liquefaction  of  the  gelatine  by  liquefying  organisms.  Care  should  be  taken 
in  preparing  the  plates  to  allow  them  to  cool  on  a  level  surface,  and  at  least  15  c.c. 

73 


74 


BACTERIA  IN  THE  AIR 


of  the  medium  should  be  employed  for  each  Petri  dish  in  order  to  ensure  an  even 
surface  and  sufficient  depth  of  medium  all  over  the  plate.  After  exposure  the 
plates  are,  under  ordinary  circumstances,  best  left  at  room  temperature  during  the 
development  of  the  colonies,  but  if  it  is  desired  to  examine  the  bacteria  alone  it  will 
be  found  well  to  favour  the  growth  of  these  at  the  expense  of  the  moulds,  by  first 
incubating  the  dish  at  a  temperature  of  37°  C.  for,  say,  eighteen  hours.  In  any 
case  the  plate  should  be  shielded  from  light,  or  otherwise  many  of  the  chromogeriic 
organisms  will  not  assume  their  typical  coloration.  Should  it  be  desired  to  photo- 
graph the  plates  in  order  to  obtain  a  permanent  record,  the  growth  should  be 
arrested,  and  the  organisms  killed  about  the  third  or  fourth  day  of  incubation.  The 
best  method  of  doing  this  is  to  reverse  the  dish,  and  to  pour  upon  a  piece  of  blotting 
paper  placed  on  the  inner  surface  of  the  lid,  which  will  now  be  undermost,  a 
sufficient  quantity  of  Formalin  to  saturate  it.  The  results  of  this  method  of 
examination  may  be  expressed  per  square  foot  per  minute,  the  area  of  the  Petri  dish 

/  22  \ 

being  calculated  f   =  (radius)2  x  —  J. 

2.  The  Flask  Method  of  Mquel. — Pasteur  was  the  first  to  analyse  air  by  the  culture 

method,  and  he  adopted  a  plan  which,  in 
principle,  is  ivashing  the  air  in  some  fluid 
culture  medium  which  will  retain  all  the 
particulate  matter,  which  may  then  be 
cultured  directly  or  sub-cultured  into  any 
favourable  medium.  Miquel  has  con- 
trived a  simple  piece  of  apparatus  for  the 
carrying  out  of  this  principle.  It  consists 
of  a  flask  with  a  central  tube  through  its 
own  neck  for  the  entrance  of  the  air.  On 
one  side  of  the  flask  is  a  tube  to  be  con- 
nected with  the  aspirator,  on  the  other 
side  of  the  flask  a  tube  through  which  to 
pour  off  the  contained  fluid  at  the  end  of 
the  process.  In  the  flask  are  placed  30  c.c. 
of  sterilised  water  (or,  indeed,  if  it  be  pre- 
ferred, sterilised  broth).  The  entrance 
tube  is  now  unplugged,  and  the  aspirator 
draws  through  a  fair  sample  of  the  air  in 
the  room  (say  ten  litres).  This  air  perforce 
passes  through  the  water,  and  by  the  exit 
tube  to  the  aspirator,  and  is  thereby 
washed,  leaving  behind  in  the  water  its 
bacteria.  The  aspiration  is  then  stopped, 
and  the  entrance  tube  closed.  The  water 
(plus  bacteria)  is  now  poured  out  into 
test-tubes  of  media  or  plated  out  on 
Petri's  dishes.  Provided  that  the  appar- 
atus has  been  absolutely  sterilised,  and 

that  only  sterilised  water  is  used,  any  colonies  developing  upon  the  Petri  dish  are 

composed  of  micro-organisms  from  the  air  examined. 

3.  The  Method  of  Hesse. — This  method  is  somewhat  akin  to  Pouchet's  aeroscope, 
but  is  in  addition  a  culture  method.     Hesse's  tube  is  50-70  cm.  long  and  3-5  cm. 
bore  throughout.     At  one  end  is  an  indiarubber  stopper  bored  for  a  glass  tube  to 
the  aspirator.     The  other  end  is  open.     Before  using,  the  tube  is  sterilised,  and  40 
or  50  c.c.  of  sterilised  gelatine  are  placed  in  it.     The  tube  is  now  rapidly  rotated 
in  a  groove  on  a  block  of  ice  or  under  a  cold-water  tap,  and  by  this  simple  means 
the  gelatine  becomes  fixed  and  forms  a  layer  inside  the  tube  throughout.     We  have 
therefore,  so  to  speak,  a  tube  of  glass  with  a  tube  of  gelatine  inside  it.    The 
apparatus  is  now  ready  for  use.     It  is  fixed  on  the1  tripod,  and  10-20  litres  of  air 
are   drawn  through,  and  the  tube  is   properly  plugged  and  incubated  at  room 
temperature.     In  two  or  three  days  the  colonies  appear  upon  the  gelatine.     They 
are  most  numerous  generally  in  the  first  part  of  the  tube.    The  disadvantages  of 


FIG.  11.— Miquel's  Flask. 


SEDGWICK'S  SUGAR  TUBE,  in  position  on  tripod, 
with  siphon. 


SMALL  HAND  CENTRIFUGE. 


[To  face  page  74. 


BACTERIOLOGICAL  EXAMINATION  OF  AIR  75 

this  process  are  that  dried  gelatine  does  not  catch  germs  like  the  broth  cultures 
of  Pasteur  or  Miquel,  and  that  many  organisms  are  carried  straight  through  the 
tube,  and  failing  to  be  deposited,  pass  out  at  the  aspirator  exit,  and  thus  are 
neither  caught  nor  counted.  The  Hesse  tube  is  generally  used  in  practice  with  a 
pump  consisting  of  two  flasks  and  a  double-way  indiarubber  tube.  The  flasks  have 
a  capacity  for  one  litre  of  water.  By  a  simple  arrangement  it  is  possible  to  secure 
syphon  action,  and  hence  measure  with  considerable  exactitude  the  amount  of  air 
passing  through  the  tube  (Plate  5). 

4.  Methods  of  Filtration. — Frankland,  Petri,  Pasteur,  Sedgwick,  and  others  have 
suggested  the  adoption  of  methods  of  filtration.  These  depend  upon  catching  the 
organisms  contained  in  the  air  by  filtering  them  through  sterilised  sand  or  sugar, 
and  then  examining  these  media  in  the  ordinary  way.  Many  different  kinds  of 
apparatus  have  been  invented.  Petri  aspirates  through  a  glass  tube  containing 
sterilised  sand,  which  after  use  is  distributed  in  Petri  dishes  and  covered  with 
gelatine.  The  principal  objection  to  this  method  is  the  presence  of  the  opaque 
particles  of  sand  in  and  under  the  gelatine.  Probably  it  was  this  which  suggested 
the  use  of  soluble  filters  like  sugar.  Pasteur  introduced  the  principle,  and  Frank- 
land  and  others  have  followed  it  out.  Sedgwick's  Tube  consists  of  a  comparatively 
small  glass  tube,  about  a  foot  long.  Half  of  it  has  a  bore  of  2*5  cm.,  and  the 
other  half  a  bore  of  -5  cm.  It  is  sterilised  at  150°  C.,  after  which  the  dry,  finely 
granulated  cane-sugar  is  inserted  in  such  a  way  as  to  occupy  an  inch  or  more  of 
the  narrow  part  of  the  tube  next  the  wide  part.  Next  to  it  is  placed  a  wool  plug, 
and  the  whole  is  again  sterilised.  After  sterilisation  an  indiarubber  tube  is  fixed 
to  the  end  of  the  narrow  portion,  and  thus  it  is  attached  to  the  aspirator.  The 
measured  quantity  (5-20  litres)  of  air  is  drawn  through,  and  any  participate  matter 


J 


FIG.  12. — Sedgwick's  Sugar-tube. 

is  caught  in  the  sugar.  Warm,  nutrient  gelatine  (10-15  c.c.)  is  now  poured  into  the 
broad  end  of  the  tube,  and  by  means  of  a  sterilised  stilette  the  sugar  is  pushed  down 
into  the  gelatine,  where  it  quickly  dissolves.  We  have  now  in  the  gelatine  all  the 
micro-organisms  in  the  air  which  has  been  drawn  through  the  tube.  After  plugging 
with  wool  at  both  ends,  the  tube  is  rolled  on  ice,  or  under  a  cold-water  tap,  in  order 
to  fix  the  gelatine  all  round  the  inner  wall  of  the  tube,  which  is  incubated  at  room 
temperature.  In  a  day  or  two  the  colonies  appear,  and  may  be  examined. 

Frankland  used  finely  powdered  sugar  and  glass  wool  as  filtering-medium,  and 
a  tube  with  two  constrictions.  After  passing  sufficient  air  through,  the  tube  is 
broken  in  halves  and  the  wool  and  sugar  are  pushed  by  means  of  a  sterile  needle 
into  liquefied  gelatine.  The  sugar  dissolves  and  the  organisms  are  distributed  in 
the  medium.  Andrewes  has  used  a  modification  of  this  method,  and  the  aspiration 
was  carried  out  with  a  large  brass  syringe  of  known  capacity,  fitted  with  a  two-way 
nozzle  and  cock,  so  that  the  requisite  number  of  syringefuls  could  be  aspirated 
without  disturbance.  * 

Various  other  methods,  including  Miquel's  filtration  method,  and  the  methods 
of  Laveran,  and  Wiirtz  and  Strauss,  have  been  used,  but  the  principal  are  those 
mentioned  above. 

In  respect  of  the  results  obtained  in  the  examination  of  air  bacteriologically,  it 
may  be  said  that  they  are  twofold.  First,  a  quantitative  result  is  obtained  by 
which  we  may  arrive  at  the  approximate  number  of  bacteria  and  moulds.  Secondly, 
the  quality  or  species  of  organisms  is  determined.  Reference  will  be  made  to  both 
these  points  in  the  pages  which  follow. 


*  Brit.  Me.il.   Jour.,  1902,  ii.,  p.  1534;  and  Report  to  London  County  Council. 

1902. 


76  BACTERIA  IN  THE  AIR 


Conditions  of  Bacterial  Contamination  of  Air 

There  are,  speaking  in  a  general  way,  four  chief  external  conditions 
affecting  the  occurrence  of  bacteria  in  air.  They  are  as  follow : — 

1.  The  presence  of  dust  and  air  pollution. 

2.  Dampness  of  surfaces. 

3.  Gravity. 

4.  Air  currents. 

1.  Dust  and  Air  Pollution. — Schwann  was  one  of  the  first  to 
point  out  that  when  a  decoction  of  meat  is  effectually  screened  from 
the  air,  or  supplied  solely  with  calcined  air,  putrefaction  does  not 
set  in.  It  is  true  that  Helmholtz  and  Pasteur  confirmed  this,  and 
greatly  added  to  our  knowledge  of  the  subject,  but  on  the  whole  it 
may  be  said  that  Schwann  originated  the  germ-theory  of  air,  and 
Lister  applied  it  in  the  treatment  of  wounds.  Lister  believed  that 
if  he  could  surround  wounds  with  filtered  air  free  from  dust  and 
particulate  or  germ  matter,  the  result  would  be  as  good  as  if  the 
wounds  were  shut  off  from  the  air  altogether. 

It  was  Tyndall  *  who  first  laid  down  the  general  principles  upon 
which  our  knowledge  of  organisms  in  the  air  is  based.  That  the 
dust  in  the  air  was  mainly  organic  matter,  living  or  dead,  was  a 
comparatively  new  truth;  that  epidemic  disease  was  not  due  to 
"  bad  air "  and  "  foul  drains,"  but  to  germs  conveyed  in  the  air,  was 
a  prophecy  as  daring  as  it  was  novel.  From  these  and  other  like 
investigations  it  came  to  be  recognised  that  putrefaction  begins  as 
soon  as  bacteria  •  from  the  air  gain  an  entrance  to  the  putrefiable 
substance,  that  it  progresses  in  direct  proportion  to  the  multiplication 
of  these  bacteria,  and  that  it  is  retarded  when  they  diminish  or  lose 
vitality. 

Tyndall  made  it  clear  that,  both  as  regards  quantity  and  quality 
of  micro-organisms  in  the  air  there  neither  is  nor  can  be  any 
uniformity.  The  degree  in  either  case  will  depend  on  air  pollution 
and  on  dust  particles.  Bacteria  may  be  conducted  on  particles  of 
dust — "the  raft  theory" — but  being  themselves  endowed  with  a 
power  of  flotation  commensurate  with  their  extreme  smalmess  and 
the  specific  lightness  of  their  composition,  dust  as  a  vehicle  is  not 
really  requisite.  Nevertheless  the  estimation  of  the  amount  of  dust 
present  in  a  sample  of  air  may  be  a  very  good  index  of  danger.  It 
is  to  Dr  Aitken  that  we  are  indebted  for  devising  a  method  by  which 
we  can  measure  dust  particles  in  the  air,  even  though  they  be 
invisible.  His  ingenious  experiments,  reported  in  the  Transactions 
of  the  Royal  Society  of  Edinburgh  (vol.  xxxv.),  have  demonstrated 
that  by  supersaturation  of  air  the  invisible  dust  particles  may  become 
visible.  As  is  now  well  known,  Dr  Aitken  believes  that  fogs,  mists, 
*  John  Tyndall,  F.R.S.,  Floating  Matter  of  the  Air,  1878. 


PLATE  6. 


AIR-PLATE  EXPOSED  IN  LABOURER'S  COTTAGE  IN  BUCKINGHAMSHIRE  (30  minutes). 

Agar  culture,  3  days  at  22°  C. 
(Grown  and  photographed  by  Swithinbank). 


[To  face  page  76. 


DUST  AND  AIR  POLLUTION  77 

and  the  like  do  not  occur  in  dust-free  air,  and  are  due  to  condensation 
of  moisture  upon  dust  particles.  And  much  the  same  has  been 
found  to  be  true  in  respect  to  dust  and  bacterial  pollution.  As  a 
rule,  when  the  former  is  abundant  the  latter  is  considerable.  Haldane 
and  Osborn  (vide  infra)  found  bacteria  most  numerous  in  a  workplace 
where  dust  was  most  abundant,  and  their  finding  was  merely  con- 
firmatory of  many  other  previous  researches.  On  the  other  hand 
it  should  be  remembered  that,  though  dust  forms  a  vehicle  for 
bacteria,  dusty  air  is  sometimes  comparatively  free  from  bacteria. 

For  the  conditions  which  affect  the  number  of  bacteria  in  the 
air  are  various.  In  open  fields,  free  from  habitations,  there  are 
fewer,  as  would  be  expected,  than  in  the  vicinity  of  manufactures, 
houses,  or  towns.  A  dry,  sandy  soil  or  a  dry  surface  of  any  kind 
will  obviously  favour  the  presence  of  organisms  in  the  air.  Frank- 
land  found  that  fewer  germs  were  present  in  the  air  in  winter  than  in 
summer,  and  that  when  the  earth  was  covered  with  snow  the  number 
was  greatly  reduced.  Miquel  and  Freudenreich  have  declared  that 
the  number  of  atmospheric  bacteria  is  greater  in  the  morning  and 
evening  between  the  hours  of  six  and  eight  than  during  the  rest  of 
the  day. 

There  is  no  numerical  standard  for  bacteria  in  the  air  as  there  is 
in  water.  In  houses  and  towns  it  would  rise  according  to  circum- 
stances, and  frequently  in  dry  weather  reach  thousands  per  cubic 
metre.  When  it  is  remembered  that  air  possesses  no  pabulum  far 
bacteria,  as  do  water  and  milk,  it  will  be  understood  that  bacteria 
do  not  live  in  the  air.  The  quality  and  quantity  of  air  organisms 
depend  entirely  upon  envtrCrTment  and  physical'iconditTOTis.  In  some 
researches  which  the  writer  made  into  the  air  of  workshops  in  Soho 
in  1896,  it  was  instructive  to  observe  that  fewer  bacteria  were  isolated 
by  Sedgwick's  sugar-tube  in  premises  which  appeared  to  the  naked 
eye  polluted  in  a  larger  degree  than  in  other  premises  apparently  less 
contaminated.  In  the  workroom  of  a  certain  skin-curer  the  air  was 
densely  impregnated  with  dust  particles  from  the  skin,  yet  scarcely 
a  single  bacterium  was  isolated.  Macfadyen  and  Lunt  have  also 
found  that  the  number  of  dust  particles  does  not  bear  any  relation  to 
the  number  of  bacteria.  They  found  that  air  containing  even 
millions  of  dust  particles  might  be  almost  germ-free.  In  the  polish- 
ing room  of  a  well-known  hat  firm,  in  which  the  air  appeared  to  the 
naked  eye  to  be  pure,  and  in  which  there  was  ample  ventilation, 
there  were  found  by  the  writer  numerous  bacteria  belonging  to  four 
or  five  species  of  saprophytes.  The  public  analyst  for  the  city  of 
Nottingham,  estimated  the  bacterial  quality  of  the  air  of  the  streets 
of  that  town  during  "the  goose  fair"  held  in  the  autumn.  He 
used  a  modification  of  Hesse's  apparatus,  in  which  the  gelatine  is 
replaced  by  glycerine.  The  air  was  slowly  drawn  through  and 


78  BACTERIA  IN  THE  AIR 

measured  in  the  usual  way.  Sterilised  water  was  then  added  to  bring 
the  glycerine  to  a  known  volume,  the  liquid  thoroughly  mixed,  and  a 
series  of  gelatine  and  agar  plates  made  with  quantities  varying  from 
O'l  to  2  c.c.  By  this  method  a  large  number  of  bacteria  were 
detected  in  this  particular  investigation,  including  Stapliyloroccus 
2iyogencs  aurcus  and  albus,  the  common  Bacillus  subtilis,  and,  appar- 
ently, B.  coli  communis*  Carnelly,  Haldane,  and  Anderson  found 
11  bacteria  per  litre  of  air  in  a  classroom  of  the  High  School  at 
Dundee  with  the  boys  at  rest.  But  when  the  boys  were  instructed 
to  stamp  on  the  floor,  and  thus  raise  the  dust,  the  number  rose  to  150 
bacteria  per  litre. 

During  a  six  years'  investigation  the  air  of  the  Mont  Souris 
Park  yielded,  according  to  Miquel,  an  average  of  455  bacteria  per 
cubic  metre.  In  the  middle  of  Paris  the  average  per  cubic  metre 
was  nearly  4000.  Flligge  accepts  100  bacteria  per  cubic  metre  as  a 
fair  average.  From  this  fact  he  estimates  that  "  a  man  during  a  life- 
time of  seventy  years  inspires  about  25,000,000  bacteria,  the  number 
contained  in  a  quarter  of  a  litre  of  fresh  milk."f  Many  authorities 
would  place  the  average  much  below  100  per  cubic  metre,  but  even 
if  we  accept  that  figure  it  is  at  once  clear  how  relatively  small  it  is. 
This  comparative  freedom  from  bacteria  is  due  to  sunlight,  rain, 
desiccation,  dilution  of  air,  moist  surfaces,  etc.  So  essentially  does 
the  bacterial  content  of  air  depend  upon  the  facility  with  which 
certain  bacteria  withstand  drying  that  Dr  Eduardo  Germano  has 
addressed  himself  first  to  drying  various  pathogenic  species  and  then 
to  mixing  the  dried  residue  with  sterilised  dust  and  observing  to 
what  degree  the  air  becomes  infected.J  The  typhoid  bacillus  appears 
to  withstand  comparatively  little  desiccation,  without  losing  its  viru- 
lence. Nevertheless  it  is  able  to  retain  vitality  in  a  semi -dried  con- 
dition, and  it  is  owing  to  this  circumstance,  in  all  probability,  that  it 
possesses  such  power  of  infection.  The  bacillus  of  diphtheria,  on  the 
other  hand,  is  capable  of  lengthened  survival  outside  the  body, 
particularly  when  surrounded  by  dust.  The  question  of  its  power  of 
resistance  to  long  drying  is  an  unsettled  point.  The  power  of 
surviving  a  drying  process  is,  according  to  Germano,  possessed  by  the 
Streptococcus  pyogcncs.  This  is  not  the  case  with  the  organisms  of 
cholera  or  plague.  Dr  Germano  classifies  bacteria,  as  a  result  of  his 
researches,  into  three  groups :  first,  those  like  the  bacilli  of  plague, 
typhoid,  and  cholera,  which  cannot  survive  drying  for  more  than  a 
few  hours ;  second,  those  like  the  bacilli  of  diphtheria  and  strepto- 
cocci, which  can  withstand  it  for  a  longer  period ;  thirdly,  those  like 
the  tubercle  bacillus,  which  can  very  readily  resist  drying  for  months 

*  Public  Health,  vol.  x.,  No.  4,  p.  130  (1898). 

f  FlUgge,  Grundriss  der  Hygiene,  1897,  pp.  161,  162. 

J  Zvitschrift  fiir  Hygiene,  vols.  xxiv.-xxvi. 


DAMP  SURFACES  79 

and  yet  retain  their  virulence.  It  will  be  obvious  that  from  these 
data  it  is  inferred  that  Groups  1  and  2  are  rarely  conveyed  by  the  air, 
whereas  Group  3  is  frequently  so  conveyed.  Miquel  has  recently 
demonstrated  that  certain  soil  bacteria  or  their  spores  can  remain 
alive  in  dried  dust  in  hermetically  sealed  tubes  for  as  long  a  time  as 
sixteen  years.  Even  at  the  end  of  that  period  such  soil  inoculated 
into  a  guinea-pig  produced  tetanus. 

The  presence  of  pathogenic  bacteria  in  the  air  is,  of  course,  a 
much  rarer  contamination  than  the  ordinary  saprophytes.  The 
tubercle  bacillus  has  been  not  infrequently  isolated  from  dry  dust 
in  consumption  hospitals,  and  in  exit  ventilating  shafts  at  Brompton 
the  bacillus  has  been  found.  From  dried  sputum,  it  has,  of  course, 
been  many  times  isolated,  even  after  months  of  desiccation.  Indeed, 
a  very  large  mass  of  experimental  evidence  attests  the  fact  that  the 
air  in  proximity  to  dried  tubercular  sputum  or  discharges  may 
contain  the  specific  bacillus  of  the  disease.  The  bacillus  of  diphtheria 
in  the  same  way,  but  in  a  lesser  degree,  may  be  isolated  from  the 
air,  and  from  the  nasal  mucous  membrane  of  nurses,  attendants,  and 
patients  in  a  ward  set  apart  for  the  treatment  of  the  disease,  and 
from  the  throats  and  nasal  mucous  membrane  of  persons  who  have 
been  in  contact  with  cases  of  the  disease.  Delalivesse,  examining 
the  air  of  wards  at  Lille,  found  that  the  contained  bacteria  varied 
more  or  less  directly  with  the  amount  of  floating  matter,  and 
depended  also  upon  the  vibration  set  up  by  persons  passing  through 
the  ward  and  the  heavy  traffic  in  granite-paved  streets  adjoining. 
B.  coli,  staphylococci,  and  streptococci,  as  well  as  B.  tuberculosis, 
were  isolated  by  this  observer.  Other  observers  have  found  B.  coli 
very  rarely  present  in  air  (Chick,  Andrewes,  etc.). 

2.  Moisture  or  Dampness  of  Surfaces. — It  is  an  interesting 
and  important  fact  that  except  under  special  circumstances  micro- 
organisms do  not  leave  moist  surfaces,  but  remain  adhering  to  them. 
A  clear  recognition  of  this  fact  is  essential  to  a  right  understanding 
of  the  pollution  of  air  by  bacteria.  They  cannot  leave  the  moist 
surface  of  fluids  either  under  evaporation  or  by  means  of  air  currents.* 
Only  when  there  is  considerable  molecular  disturbance,  such  as 
splashing,  can  microbes  be  transmitted  to  the  surrounding  air. 
This  is  the  reason  why  sewer  gas  and  all  air  contained  within  moist 
perimeters  is  almost  germ-free,  whereas  from  dry  surfaces  the  least 
air  current  is  able  to  raise  countless  numbers  of  organisms. 

This  principle  has  been  admirably  illustrated  in  investigations 
made  upon  expired  and  inspired  air.  In  a  report  to  the  Smithsonian 
Institute  of  Washington  (1895)  upon  the  composition  of  expired  air, 

*  Fliigge  has  lately  attempted  to  demonstrate  that  an  air  current  having  a 
velocity  of  four  metres  per  second  can  remove  bacteria  from  surfaces  of  liquids  by 
detaching  drops  of  the  liquid  itself. 


80  BACTERIA  IN  THE  AIR 

it  is  concluded  that  "in  ordinary  quiet  respiration  no  bacteria 
epithelial  scabs  or  particles  of  dead  tissue  are  contained  in  the 
expired  air.  In  the  act  of  coughing  or  sneezing  such  organisms  or 
particles  may  probably  be  thrown  out."  The  mucous  membrane 
lining  the  cavity  of  the  mouth  and  respiratory  tract  is  a  moist 
perimeter,  from  the  walls  of  which  no  organisms  can  rise  except 
under  molecular  disturbance.  The  popular  idea  that  bacteria  can 
be  "given  off  by  the  breath"  is  therefore  contrary  to  the  laws  of 
organismal  pollution  of  air.  The  required  conditions  are  not 
fulfilled,  and  such  breath  infection  must  be  of  extremely  rare 
occurrence  except  in  speaking,  spitting,  sneezing  or  coughing 
(Fltigge).  Air  can  only  become  infective  when  impregnated  with 
organisms  arising  from  dried  surfaces. 

Another  series  of  investigations  were  conducted  by  Drs  Hewlett 
and  St  Glair  Thomson,  and  dealt  with  the  fate  of  micro-organisms 
in  inspired  air  and  micro-organisms  in  the  healthy  nose.  They 
estimated  that  from  1500  to  14,000  bacteria  were  inspired  every 
hour.  Yet,  as  we  have  seen,  expired  air  contains  practically  none 
at  all.  It  is  clear,  then,  that  the  inspired  bacteria  are  detained  some- 
where. Lister  has  pointed  out,  from  observation  on  a  pneumo-thorax 
caused  by  a  wound  of  the  lung  by  a  fractured  rib,  that  bacteria  may 
be  arrested  before  they  reach  the  air  cells  of  the  lung,  and  other 
observations  confirm  this  fact,  although  of  course  there  are  several 
well-known  exceptions  (e.g.  tubercle  of  the  lung).  Hence  it  is  at 
some  intermediate  stage  that  they  are  detained.  Hewlett  and 
Thomson  examined  the  mucus  from  the  wall  of  the  trachea,  and 
found  it  germ-free.  It  was  only  when  they  examined  the  mucous  mem- 
brane and  moist  vestibules  and  vibrissae  of  the  nose  that  (they  found 
bacteria*  Here  they  were  present  in  abundance.  The  ciliated 
epithelium,  the  mucus,  and  the  bactericidal  influence  of  the  wandering 
or  "  phagocyte  "  cells,  probably  all  contribute  to  their  final  removal.* 

There  can  be  no  doubt  that  the  large  number  of  bacteria  present 
in  the  moist  surfaces  .of  the  mouth  is  the  cause  of  a  variety  of 
ailments,  and  under  certain  conditions  of  ill-health  organisms  may 
through  this  channel  infect  the  whole  body.  Dental  caries  will  occur 
to  everyone's  mind  as  a  disease  probably  due  in  part  to  bacteria.  As 
a  matter  of  fact,  acids  (due  to  acid  secretion  and  acid  fermentation) 
and  micro-organisms  are  two  of  the  chief  causes  of  decay  of  teeth. 
Defects  in  the  enamel,  inherent  or  due  to  injury,  retention  of  debris 
on  and  around  the  teeth,  and  certain  pathological  conditions  of  the 
secretion  of  the  mouth,  are  predisposing  causes,  which  afford  a 

*  Hewlett  and  Thomson  graphically  demonstrated  the  bactericidal  power  of  the 
nasal  mucous  membrane  by  noting  the  early  removal  of  Bacillus  prodigiosus, 
which  had  been  purposely  placed  on  the  healthy  Schneiderian  membrane  of  the 
nose. 


BACILLUS  COLI  49 

General  Note. — Whilst  the  above  description  applies  to  the 
normal  type  of  B.  coli,  it  should  be  clearly  understood  that  a  large 
number  of  bacilli  have  been  described  which  possess  some,  but  not 
all,  of  the  above  characters.  Eefik  has  described  (Ann.  de  VInst. 
Pasteur,  x.,  1896,  242),  five  varying  types  very  similar  to  the  normal 
B.  coli,  but  differing  in  one  or  more  characters.  Almost  all  forms, 
however,  have  some  features  in  common,  e.g.,  motility,  few  flagella, 
and  characteristic  growth  on  potato.  Moreover,  there  are  a  group 
of  organisms  allied  to  B.  coli,  and  often  associated  with  it.  Like  it 
also,  they  are  related,  etiologically  or  otherwise,  to  similar  pathological 
processes.  Kefik's  types  are  briefly  as  follows : — 

A.  Ferments  lactose,  coagulates  milk,  but  gives  no  indol  reaction. 

B.  Ferments  lactose,  does  not  coagulate  milk,  gives  indol  reaction. 

C.  Ferments  lactose,  does  not  coagulate  milk,  does  not  give  indol 
reaction. 

D.  Does  not  ferment  lactose,  coagulates  milk,  does  not  give  indol 
reaction. 

E.i  Does  not  ferment  lactose,  does  not  coagulate  milk,  does  not 
give  indol  reaction. 

Mervyn  Gordon  has  made  a  careful  study  of  the  B.  coli  and  its 
allies  which  he  classified  according  to  their  reactions  and  their  flagella. 
He  differentiated  16  varieties.*  Horrocks  studied  the  cultural  char- 
acters of  150  "varieties"  of  B.  coli  isolated  partly  from  normal  and 
partly  from  typhoid  stools,  f  Other  workers  have  observed  an 
enormous  variety  of  minor  differences.  The  important  point  is  the 
diagnosis  of  B.  coli,  and  the  following  characters  are  now  chiefly  relied 
upon  (see  also  p.  472).  1.  The  B.  coli  group  is  non-sporing  and  non- 
liquefying;  2.  The  members  of  the  group  rarely  stain  by  Gram's 
method ;  3.  They  produce  acid  and  gas  with  both  glucose  and  lactose ; 
4.  They  produce  acid  in  milk  and  they  usually  also  coagulate  it ;  5. 
They  produce  acid  and  gas  in  bile-salt-glucose  broth ;  6.  They  grow 
well  at  a  temperature  of  42°  C.J  Other  fairly  reliable  features  are 
motility,  a  small  number  of  flagella,  a  fairly  typical  growth  on  potato, 
and  more  rapid  development  on  all  media  than  the  typhoid  bacillus. 
But  there  is  not  at  the  present  time  a  complete  unanimity  of  opinion 
as  to  the  most  reliable  characters  for  diagnostic  purposes.  § 

*  Jour,  of  Path.  andBact.,  1897,  vol.  iv.,  p.  438. 

f  Bacteriological  Examination  of  Water,  1901,  p.  94;  Jour,  of  Hyg.,  1901,  p.  202. 

J  Roy.  Com.  on  Sewage  Disposal,  Second  Report,  1902,  p.  101.  See  also  Brit. 
Med.  Jour.,  1903,  i.  418  (Klein),  for  summary  of  characters  of  B.  coli. 

§  Houston  considers  the  following  the  most  useful  tests  for  B.  coli:  (1)  Gas 
formation  in  ordinary  gelatine  "shake"  cultures;  (2)  indol  in  broth  cultures; 
(3)  acid  and  clot  in  litmus  milk  -  cultures ;  (4)  greenish-yellow  fluorescence  in 
neutral-red  broth  cultures  ;  (5)  gas  and  acid  in  lactose-peptone  cultures ;  (6)  gas, 
acid,  and  clot  in  peptone-lactose  milk  cultures  ;  (7)  gas  and  acid  in  glucose-peptone 
cultures;  (8)  reduction  of  nitrate  to  nitrite  in  nitrate  broth  cultures;  (9)  strong 
acid  in  Proskauer  and  Capaldi's  medium  No.  1,  and  no  definite  production  of  acid 

D 


50  BACTERIA  IN  WATER 

The  significance  of  B.  coli  is  of  course  its  potential  pathogenicity, 
and  its  similarity  to  the  typhoid  bacillus,  but  above  all  its  relation 
to  sewage.  Roux,  Rodet,  and  others  have  stated  that  B.  coli,  under 
certain  circumstances,  may  assume  a  character  not  distinguishable 
from  B.  typhosus,  both  in  its  biological  and  cultural  characteristics 
and  in  its  pathogenic  properties.  Chantemesse,  Widal,  and  others 
have  held  that  polluted  waters  owe  their  power  to  produce  typhoid 
fever  to  the  presence  of  B.  coli,  and  that  possibly  the  organisms 
are  transformable  the  one  into  the  other.  Klein  and  many  other 
bacteriologists,  as  the  result  of  very  numerous  experiments,  have 
been  unable  to  effect  any  transformation  of  one  form  into  the 
other.  Each  organism  has  retained  unimpaired  its  differential 
characters. 

Certain  strains  of  B.  coli  are  distinctly  pathogenic  for  lower 
animals,  and  there  is  some  ground  for  considering  the  organism  a 
cause  of  disease  (epidemic  diarrhoea  and  other  conditions)  in  man, 
either  by  itself  or  in  association  with  other  organisms  (Delepine). 
In  the  third  place,  as  is  pointed  out  elsewhere,  B.  coli  is  a  sewage 
organism,  and  the  chief  importance  of  its  detection  in  water  is  an 
indication  of  sewage  pollution  and  therefore  of  possible  contamination 
of  the  water  with  specific  bacteria.  It  is  therefore  a  most  reliable 
test  of  pollution.  Klein  and  Houston  have  emphasised  the  importance 
of  the  presence  of  B.  coli  and  the  B.  cnteritides  sporogencs  in  water  as 
indication  of  sewage  pollution,  and  by  this  means  a  demonstration  of 
the  presence  of  sewage  in  ~  water  can  be  carried  to  an  incomparably 
higher  degree  than  by  chemical  examination.  Chemistry  is  powerless 
to  detect  pollution '  by  pathogenic  germs  or  the  small  amount  of 
organic  pollution  which  can  be  detected  by  bacteriology,  which  is  ten 

in  Proskauer  and  Capaldi's  medium  No.  2 ;  (10)  presence  of  motility ;  (11) 
non-liquefaction  of  gelatine  ;  and  (12)  acidity  in  litmus  whey  cultures,  varying  from 

about  20-40  c.c.  —  Na2CO3  per   100  c.c.   of  culture.     In   dealing  with  sewage, 

effluents,  and  non-drinking-water  streams,  Houston  employs  the  first  three  tests, 
but  in  dealing  with  drinking-water,  the  first  five  tests  (Fourth  Report  of  Royal 
Commission  Sewage  Disposal,  1904,  p.  106). 

McWeeney  relies  chiefly  upon  '(a)  the  character  of  gelatin  colony  and  non- 
liquefaction  of  that  medium,  even  after  a  long  time  ;  (6)  non-retention  of  Gram's 
stain ;  (c)  fermentation  of  lactose  with  gas  and  acid  formation  ;  (d)  coagulation  of 
milk  within  four  days  at  37°  C.  ;  (e)  production  of  yellowish-green  fluorescence  in 
neutral-red-agar-shake  culture ;  and  (/)  production  of  indol  in  liquid  peptone  media. 
(Report  of  Local  Government  Board  for  Ireland,  1904).  Klein  describes  B.  coli 
as  a  motile,  non-spore-bearing  bacillus,  possessing  a  limited  number  of  flagella, 
capable  of  fermenting  glucose  and  lactose,  of  curdling  milk  with  the  production  of 
acid,  of  forming  indol  in  broth  culture,  reducing  neutral  red  with  the  production  of 
a  green  fluorescence^producing  gas-bubbles  in  nutrient  jelly,  of  forming  a  more  or 
less  brownish  growth  ran  steamed  potato,  and  of  producing  on  the  surface  of  gelatin 
a  dry,  translucent  growth  which  does  not  liquefy  the  gelatin.  The  bacilli,  under  the 
microscope,  appear  asfcylindrical  rods,  showing  more  or  less  pronounced  motility, 
and  they  do  not  stainjby  the  method  of  Gram  (see  also  Appendix,  pp.  466  and  472). 


Bacillus  coli  communis.     Surface  gelatine  plate  culture,  O'l  c.c.  ot  ^^  c.c.  of  Rugby  sewage. 


GAS  IN  GELATINE  SHAKE  CULTURE,  24  hours  at  20°  C. 
From  left  to  right  the  tubes  represent  TJn,  Tn'rtiT,  TBJTO>  Tn^nr>  c.c.  of  Nottingham  crude  sewage. 

[To  face  page  50. 


BACILLUS  COLI  51 

to  one  hundred  times  less  than  that  detectable  by  chemistry.*  It  is, 
however,  important  to  bear  in  inind  that  something  more  than  the 
mere  presence  of  B.  coli  must  be  ascertained.  The  comparative 
numbers  present,  the  relative  abundance,  and  the  general  character 
and  source  of  the  water  must  be  considered.  Waters  containing  no 
B.  coli  in  100  c.c.  are  of  course  of  a  high  degree  of  purity.f  In 
upland  surface  waters  the  presence  of  B.  coli  in  such  a  small  amount 
as  1  c.c.,  may  be  sufficient  to  condemn  the  waters.  Certainly  drinking- 
water  from  a  deep  well  should  contain  no  B.  coli.  The  presence  in 
a  water  of  B.  coli  in  conjunction  with  streptococci  or  even  the  spores 
of  B.  enteritidis  sporogenes,  or  both,  would  of  course  indicate  serious 
pollution. 

The  differential  diagnosis  of  B.  coli  from  its  allies  or  other 
organisms  is  not  always  a  simple  matter.  An  adherence  to  the 
characteristics  set  out  above  will  generally  prove  safe  guidance,  but 
reliance  should  not  be  placed  upon  any  single  character  or  test. 
The  tendency  to  adopt  some  rapid  and  easily-applied  test  for  this 
organism  is  strongly  to  be  deprecated,  as  likely  to  lead  to  error. 
Nothing  can  take  the  place  of  the  careful  study  and  sub-culture  of 
the  suspected  organism  in  this  and  in  all  other  species.  At  the  same 
time,  it  has  been  found  that  diagnostic  aid  is  obtained  by  a 
comparison  of  some  of  the  biological  characters  of  the  colon  and 
allied  groups  of  bacteria.  They  may  be  divided  into  four  divisions  :— 
(1)  The  proteus  group,  the  members  of  which  are  motile,  liquefy 
gelatine,  produce  gas  in  glucose  and  sucrose  but  not  in  lactose,  curdle 
and  acidulate  milk  very  slowly,  and  usually  produce  indol ;  (2)  the  coli 
group  include  motile  bacilli,  producing  gas  in  glucose  and  lactose, 
curdle  milk  rapidly,  nearly  always  produce  indol,  but  do  not  liquefy 
gelatine,  and  do  not  retain  Gram's  stain;  (3)  the  group  including 
B.  lactis  cerogenes  are  non-motile  bacilli,  which  do  not  liquefy  gelatine 
but  which  curdle  and  acidulate  milk  and  ferment  sugars  other  than, 
glucose;  and  (4)  the  enteritidis  group  contain  bacilli  which  are 
motile,  which  only  ferment  glucose,  and  which  do  not  liquefy  gelatine 
or  curdle  milk,  which  is  ultimately  rendered  alkaline.  This  group 
includes  B.  enteritidis  of  Gaertner,  the  para-colon  and  the  para- 
typhoid bacilli. 

Streptococci  in  Water. — Houston  considers  the  presence  of  strepto- 
cocci in  water  as  indication  of  recent  and  dangerous  pollution  of 
water.  They  are  absent  even  in  large  quantities  of  pure  water  and 
in  virgin  soils.j  Streptococci,  as  a  class,  are  delicate  germs  that 
readily  lose  their  vitality  and  die  when  the  physical  conditions  are 
unfavourable,  and  they  comprise  species  highly  pathogenic  to  human 

*  Medical  Supplement  to  Report  of  Local  Government  Board,  1898-99,  p.  498. 

t  See  also  Jour,  of  Hyg.,  1902,  p.  339  (Savage). 

J  Report  of  Local  Government  Board,  1899-1900,  p.  -183. 


52  BACTERIA  IN  WATER 

beings.  They  are  present  in  human  faeces  and  in  crude  sewage  in 
considerable  number;  and  as  we  have  said,  they  are  absent  from 
relatively  large  amounts  of  pure  waters  and  virgin  soils,  but  present 
in  abundance  in  water  and  soil  recently  polluted  with  animal 
dejecta.  It  is  not  claimed  that  all  streptococci  are  necessarily  delicate 
germs,  or  pathogenic,  or  of  recent  animal  outcome.  It  may  be  that 
certain  streptococci  are  comparatively  hardy  germs,  and  that  others 
may  be  capable  of  multiplying  in  Nature  outside  the  animal  body. 
Again,  there  may  be  streptococci  in  Nature  which  do  not  owe  their 
origin  to  excremental  matter,  and  doubtless  many  of  them  may  be 
non-pathogenic,  although  this  latter  circumstance  is  no  proof  that 
at  a  stage  prior  to  their  isolation  they  were  non-virulent,  nor  does 
it  impair  the  value  of  the  test  as  an  indication  of  recent  fouling  with 
objectionable  matters. 

Houston  found  streptococci  habitually  present  in  crude  sewage 
in  TTjVo  c-cv  present  in  human  faeces  in  one  milligramme,  and  present 
in  minimal  quantities  of  soils  and  water  recently  polluted  with 
matters  of  animal  outcome.  These  results  encourage  the  belief  that 
the  streptococcus  test  is  one  of  the  most  delicate  yet  suggested  for 
detecting  recent,  and  therefore,  presumably,  specially  dangerous, 
pollution. 

The  question  of  relative  abundance  in  connection  with  the  strepto- 
coccus test  also  deserves  consideration.  For  if  streptococci  are  absent 
from  10  c.c.  or  more  of  pure  waters  and  present  in  TTroo-  c-c-  °f 
crude  sewage  the  distinctions  as  regards  streptococci  between  water 
and  sewage  is  sufficiently  great  to  allow  of  considerable  latitude  being 
observed  in  framing  a  standard  without  seriously  impairing  the  value 
of  the  test.  What  standard  should  be  adopted  is  a  matter  of  opinion, 
but  as  a  rule  it  may  be  said  that  the  presence  of  streptococci  are  to 
be  thought  of  as  indicating  extremely  recent,  and  B.  coli  less  recent, 
but  still  not  remote,  pollution  of  animal  sort  (Houston).  The 
presence  of  B.  entcritidis  sporogenes,  however,  cannot  be  considered  to 
afford  evidence  of  pollution  bearing  a  necessary  relation  to  the  recent 
evacuations  of  animals.  Streptococci  and  B.  coli  are  either  altogether 
absent  or  present  in  sparse  amount  in  virgin  soils,  and  may  be 
absent  even  from  polluted  soils,  unless  the  contamination  is  of 
comparatively  recent  sort.  In  soils  recently  polluted  with  animal 
matters  streptococci  and  B.  coli  are  of  course  present  in  abundance. 
B.  enteritidis  sporogenes  may  be  present  even  in  seemingly  virgin  soils, 
but  in  sparse  proportion  compared  with  the  large  number  found  in 
cultivated  and  polluted  soils.  Lastly,  the  presence  of  streptococci 
in  any  number  in  a  water  supply  points  not  only  to  recent  animal 
pollution,  but  also  implies  that  the  antecedent  conditions — condi- 
tions intervening  between  the  period  of  pollution  of  the  water  and  the 
time  of  collection  of  the  sample — could  hardly  have  been  of  so  un- 


PATHOGENIC  BACTERIA  IN  WATER  53 

favourable  a  character  as  to  destroy  the  vitality  of  seemingly  more 
hardy  microbes — for  example,  the  typhoid  bacillus.  The  same 
cannot  be  said  for  the  B.  coli  test,  since  B.  coli  is  a  more  hardy  germ 
than  B.  typhosus. 

Broadly,  therefore,  it  will  be  seen  that  the  presence  of  B.  coli  or 
B.  enteritidis  sporogenes  or  Streptococci  in  a  water  is  presumptive 
evidence  of  sewage  pollution.  But  that  in  forming  an  opinion  it  is 
essential  to  bear  in  mind  the  relative  abundance  of  organisms  per 
c.c.  and  the  relative  abundance  of  certain  species. 

(c)  Pathogenic  Bacteria  in  Water. — The  two  chief  types  of 
disease-producing  organisms  found  in  water  are  the  bacillus  of 
typhoid  fever  and  the  bacillus  of  cholera.  These  diseases  and  their 
causal  organisms  are  dealt  with  subsequently  (see  pp.  298  and  384). 
Here  it  will  only  be  necessary  to  note  one  or  two  general  facts  as  to 
the  relation  of  pathogenic  organisms  to  water  supplies. 

In  sterilised  water,  and  in  very  highly  polluted  water  or  sewage, 
pathogenic  bacteria  do  not  flourish.  In  the  former  case  they  die  of 
starvation,  although  there  are  experiments  on  record  which  appear 
not  to  support  this  view ;  in  the  latter  case  they  are  killed  by  the 
enormous  competition  of  common  bacteria.  Even  in  ordinary  water 
there  is  a  wide  divergence  of  behaviour.  Some  bacteria  are  destroyed 
in  a  few  hours ;  others  appear  to  flourish  for  weeks.  In  all  cases  the 
spores  are  able  to  resist  whatever  injurious  properties  the  water  may 
have  much  more  persistently  than  the  bacilli  themselves.  These 
changes  in  the  vitality  of  bacteria  in  water,  partly  due  to  the  water 
and  partly  to  the  other  micro-organisms,  bring  about  two  character- 
istics which  it  is  important  to  remember,  viz.,  that  pathogenic  germs 
in  water  are,  as  a  rule,  scanty  and  intermittent.  It  is  these  features 
in  conjunction  with  the  enormous  quantities  of  common  water  bacteria 
which  make  the  search  for  the  bacillus  of  typhoid  fever  what  Klein  has 
called  "  searching  for  a  needle  in  a  rick  of  hay."  Not  that  it  cannot 
be  detected,  but  its  detection  is  one  of  the  most  difficult  of  investiga- 
tions. In  recent  years  the  typhoid  bacillus  has  been  isolated  from 
water  which  had  given  rise  to  cases  of  typhoid  fever  at  Pierrefonds 
(Widal  &  Chantemesse),  Dijon  (Vaillard),  Chateaudun,  Cuxhaven 
(Dun bar),  and  possibly  one  or  two  other  instances.*  Undoubtedly 
a  large  number  of  epidemics  have  been  due  to  typhoid  infected  water, 
but  for  obvious  reasons  (long  incubation  of  typhoid,  the  fact  that 
the  bacillus  only  lives  in  water  for  a  few  days,  etc.),  the  cases  where 
the  bacillus  has  been  actually  isolated  are  very  few.  In  the  Milroy 
Lectures  for  1902,  Professor  Corfield  gives  records  of  between  50  and 
GO  typhoid  epidemics  since  1864.  We  shall  refer  to  this  matter 
subsequently  when  Bacillus  typhosus  is  under  consideration. 

In  artificial  cultivation  water  bacteria  respond  very  readily  to 
*  Brit.  Med.  Jour.,  1900,  ii.  p.  1198. 


54  BACTERIA  IN  WATER 

external  conditions.  Increase  of  alkalinity  ("01  grams  of  sodium 
carbonate  added  to  10  c.c.  of  ordinary  gelatine)  causes  the  number 
of  colonies  to  be  five  or  six  times  greater  than  that  revealed  by  using 
ordinary  gelatine ;  on  the  other  hand,  very  slightly  increasing  the 
acidity  of  a  medium  as  markedly  diminishes  the  number  of  bacteria. 
Advantage  is  taken  of  this  in  culturing  the  bacillus  of  typhoid,  which 
is  not  inhibited  by  an  acid  medium. 

Water  may  become  contaminated  with  pathogenic  bacteria  in  a 
variety  of  ways,  as  pollutions  at  the  source,  in  the  course,  and  at  the 
periphery.  Gathering  grounds  are  frequently  the  source  of  the 
pollution.  The  Maidstone  typhoid  epidemic  was  an  example.  Here 
some  of  the  springs  supplying  the  town  with  water  were  con- 
taminated by  several  typhoid  patients.  Frequently  on  the  gathering 
ground  one  may  find  a  number  of  houses  the  waste  and  refuse  of 
which  will  furnish  ample  surface  pollution,  which  in  its  turn  may 
readily  pass  into  a  collecting  reservoir  or  a  well.  On  one  occa- 
sion the  writer  investigated  the  cause  of  typhoid  fever  in  a  large 
country  house  in  Oxfordshire,  and  traced  it  to  pollution  of  the 
private  well  by  surface  washings  from  the  stable  quarters.  Leak- 
age of  house  drains  into  wells  is  not  an  infrequent  source  of 
contamination. 

The  same  cause  is  generally  operative  in  cases  of  pollution  of  a 
water  supply  in  its  course  from  the  source  to  the  cisterns  or  taps 
at  the  periphery,  viz.,  a  sewer  or  drain  leaking  into  the  water  supply. 
Water  companies  and  those  responsible  for  water  supply  appeal- 
frequently  to  hold  the  opinion  that  so  long  as  there  is  sand  filtration 
or  subsidence  reservoirs,  it  is  unnecessary  to  consider  the  gathering 
ground  or  possible  contamination  during  transit.  But  it  happens 
that  a  frost  may  completely  dislocate  the  efficient  action  of  a  filter, 
and  times  of  flood  may  prevent  proper  sedimentation ;  then  our 
dependence  for  pure  water  is  wholly  upon  the  gathering  ground  and 
source.  Hence  we  find  water  contaminated  at  its  source  by  polluted 
wells,  by  sewage-infected  rivers  and  streams,  by  drainage  of  manured 
fields,  by  innumerable  excremental  pollutions  over  the  areas  of  the 
gathering  grounds,  and  in  transit  by  careless  laying,  bad  construction 
and  jointing  of  pipes,  and  close  proximity  of  such  drain  pipes  to  the 
water  supply. 

In  the  third  place,  we  may  get  a  water  infected  at  the  periphery, 
in  the  house  itself.  Such  cases  are  generally  due  to  two  causes: 
filthy  cisterns  and  pipes  or  suction.  Cisterns  per  se  are  more  or  less 
indispensable  where  a  constant  service  does  not  exist,  but  they  should 
be  inspected  from  time  to  time  and  maintained  in  a  cleanly  condition. 
Suction  into  the  tap  has  been  emphasised  by  Dr  Vivian  Poore  as 
a  cause  of  pollution.  It  is  liable  to  occur  whenever  a  tap  is  left 
turned  on,  and  a  vacuum  is  produced  in  the  supply  pipe  by  inter- 


INTERPRETATIONS  OF  BACTERIOLOGY  55 

mission  of  the  water  supply,  so  that  foul  gas  or  liquid  is  sucked  back 
into  the  house-pipe. 

A  further  point  has  relation  to  bacterially  polluted  water  when  it 
has  gained  entrance  to  the  body.  It  has  been  known  for  some  time 
past  that  not  all  waters  polluted  with  disease  germs  produce  disease. 
As  we  have  before  said,  this  depends  upon  the  infective  agent,  its 
quantity  and  quality,  and  upon  the  human  body.  The  body  is  able 
in  many  cases  to  resist  a  small  dose  of  poison.  It  is,  however, 
necessary  to  infection,  especially  in  water-borne  disease,  that  the 
tissues  shall  be  in  some  degree  disordered,  weakened,  or  injured.  For 
instance,  the  perverted  action  of  the  stomach  influences  the  acid 
secretion  of  the  gastric  juice,  through  which  bacilli  might  then  pass 
uninjured.  Particularly  must  this  be  so  in  the  bacillus  of  cholera, 
which  is  readily  killed  by  the  normal  acid  reaction  of  the  stomach. 
Hence,  in  this  disease  at  least,  it  is  the  opinion  of  bacteriologists  that 
the  condition  of  the  mucous  membrane  of  the  stomach  is  of  primary 
importance.  Metchnikoff  has  indeed  demonstrated  the  presence  of 
the  bacillus  of  cholera  in  the  intestinal  excretion  of  apparently  healthy 
persons,  which  shows  that  they  were  protected  by  the  resistance  of 
their  tissues  to  the  bacilli.  Further  light  has  been  thrown  on  this 
question  by  the  researches  of  MacFadyen,  who  has  pointed  out  that 
suspensions  of  cholera  bacilli  in  water  passed  through  the  stomach 
untouched,  and  were  thus  able  to  exert  their  evil  influence  in  other 
parts  of  the  alimentary  canal.  When,  however,  cholera  bacilli  were 
suspended  in  milk,  none  appeared  to  escape  the  germicidal  action 
of  the  gastric  juice.  The  explanation  of  this  is  probably  the  simple 
one  that  the  stomach  reacted  with  its  secretion  of  gastric  juice  only 
to  food  (milk),  but  passed  the  water  on  into  the  lower  and  more 
absorptive  parts  of  the  alimentary  canal.  Such  a  condition  of  affairs 
clearly  increases  the  danger  due  to  water-borne  germs. 

The  Interpretation  of  the  Finding's  of  Bacteriology 

Bacteriology  is  the  most  direct  and  delicate  test  of  the  safety  of  a 
water  for  drinking  purposes.  By  it  we  obtain  exact  information  not 
alone  as  to  the  constitution  of  a  water,  but  as  to  its  potentiality  to 
cause  disease.  It  is  also  a  more  delicate  test  than  a  chemical 
examination.*  Klein  and  others  have  shown  that  by  bacteriological 
methods  it  is  possible  to  detect  smaller  degrees  of  sewage  pollution 
than  by  chemistry.  On  the  other  hand,  it  is  useless  to  expect  to 
learn  of  the  exact  chemical  constitution  of  a  water  by  bacteriological 
methods.  Bacteriology  must  be  interpreted  by  what  it  can 

*  Clark  and  Gage  state  that  polluted  waters  which  might  become  unfit  for 
drinking  purposes  are  more  plainly  indicated  by  a  single  chemical  analysis  than  by 
a  single  determination  of  B.  coli. 


56  BACTERIA  IN  WATER 

do  and  not  by  what  it  cannot;  and  in  a  general  way  it  may 
be  said  that  there  are  three  groups  of  facts  contained  in  a 
systematic  bacteriological  report  of  water.  These  findings  are 
concerned  with  the  number  of  bacteria  per  c.c.,  the  presence  of 
any  organisms  of  contamination,  and  the  presence  of  any  specific 
organisms  of  disease. 

(1)  Number  of  Bacteria  per  ex. — It  would  appear  that  in  the  past 
a  great  deal  too  much  weight  has  been  attributed  to  the  number  of 
bacteria  per  c.c.  This  fact  is  not  of  the  first  importance  for  two 
obvious  reasons.  In  the  first  place  there  is  no  standard  as  to  how 
many  bacteria  should  be  present  in  1  c.c.  of  a  potable  water,  and  in 
the  second  place  there  is  no  known  means  by  which  this  number 
can  be  accurately  measured.  In  this  country  any  number  of  bacteria 
under  one  hundred  per  c.c.  is  generally  considered  low.  The 
metropolitan  water  supply,  as  consumed,  usually  contains  less  than 
twenty  bacteria  per  c.c.  Deep  -  well  waters  and  spring  waters 
frequently  contain  very  few  bacteria.  Polluted  or  surface  waters 
contain  thousands  of  organisms  per  c.c.  More  than  this,  no 
standard  exists.  Nor  would  any  numerical  standard  taken  alone 
be  of  much  value,  for  the  reason  that  the  number  of  bacteria  in 
water  is  of  comparatively  little  value  apart  from  a  knowledge  of  the 
species,  and  moreover  a  really  accurate  record  of  the  number  of 
bacteria  per  c.c.  is  not  obtainable.  Whether  the  organisms  detected 
be  many  or  few  depends  upon  a  variety  of  external  circumstances, 
such  as  medium  used  for  cultivation,  temperature  and  period  of 
incubation,  length  of  time  of  cultivation  before  counting,  or  the 
use  or  not  of  a  lens  when  counting.  For  these  reasons  it  is 
evident  that  great  reliance  cannot  be  placed  upon  the  number 
of  bacteria  per  c.c.  returned  in  bacteriological  reports,  and  it  is 
well  that  should  be  understood.  The  only  circumstances  under 
which  such  returns  are  valuable  are  (a)  when  used  in  a  series  of 
examinations  of  the  same  water  supply,  when  such  returns,  if  always 
obtained  under  the  same  conditions,  are  of  great  comparable  value, 
and  (&)  when  used  in  the  examination  of  water  before  and  after 
filtration.  In  these  two  circumstances  the  number  of  organisms  per 
c.c.  is  of  great  value  in  forming  an  opinion  as  to  pollution  or  as  to 
failure  of  filtration. 

(2)  Presence  of  Organisms,  of  Contamination. — In  the  general 
bacteriological  examination  of  water  this  point  is  perhaps  the  most 
important.  Judgment  must  be  formed  on  two  facts,  namely,  the 
presence  of  any  of  the  "bacteria  of  indication,"  such  as  B.  coli, 
B.  enteritidis  sporogenes,  streptococci,  and  the  para-colon  types 
(enteritidis,  Gaertner,  and  the  chologenes  type),  and  the  relative 
abundance  of  these  species.  The  latter  point  is  one  of  importance. 
The  chief  organism  of  indication  is  B.  coli,  including  under  that 


ORGANISMS  OF  CONTAMINATION  57 

term  the  typical  bacillus  and  closely  allied  organisms.  When 
this  bacillus  can  be  detected  in  a  small  measured  quantity  of 
water,  that  is  to  say,  in  1,  2  or  3  c.c.,  it  is  assumed  (a)  that  the 
organism  has  gained  access  to  the  water  from  sewage,  and  (I)  that 
recently,  (c)  It  is  further  assumed  that  certain  disease-producing 
bacteria  which  occur  frequently  in  sewage  may  also  be  present  in 
the  water,  though  if  present  at  all  in  the  water,  in  considerably 
smaller  numbers  than  B.  coli.  (d)  Further,  judging  the  matter 
broadly,  the  higher  the  number  of  B.  coli  the  heavier  will  have  been 
the  recent  sewage  pollution,  and  the  greater  the  probability  of  the 
presence  of  disease-producing  bacteria.  Conversely,  if  B.  coli  is 
not  present,  one  may  assume  with  some  probability  of  being  correct, 
that  such  disease-producing  bacteria  as  the  bacillus  of  typhoid  fever 
will  also  be  absent,  and  that  the  particular  sample  of  water  under 
examination  might  safely  be  used  for  drinking  purposes. 

There  is  difference  of  opinion  as  to  the  exact  quantity  of  a  water 
which  must  be  free  from  a  single  specimen  of  B  coli  in  order  that  it 
may  be  said  that  the  sample  is  a  "  safe "  one ;  but  many  would  in 
practice  accept  the  standard  1  or  2  c.c. 

It  has  already  been  stated  that  the  presence  of  B.  coli  in  a  water  is 
not  of  importance,  because  this  organism  itself,  under  the  ordinary 
conditions,  is  likely  to  be  harmful,  but  rather  because  it  serves  as  an 
index  of  sewage  or  surface  pollution.  In  this  connection  it  may  be 
said  that  a  single  examination  of  a  water  is  of  practically  no  value 
when  the  results  of  the  bacteriological  examination  are  favourable ; 
it  is  only  after  repeated  examination  has  shown  that  B.  coli  'is  absent 
from  the  water  for  a  prolonged  period,  and  after  local  inspection  has 
shown  that  there  are  no  possible  sources  of  dangerous  sewage  con- 
tamination, that  one  is  justified  in  giving  a  positive  opinion  as  to 
the  safety  of  a  water.  On  the  other  hand,  a  single  bacteriological 
examination  with  an  unfavourable  result  will  prove  the  actual 
occurrence,  and  suggest  the  possible  recurrence,  of  sewage  contamina- 
tion, and  will  necessitate  renewed  inspection  if  no  obvious  source  of 
contamination  is  known  to  exist. 

B.  coli  is  commonly  considered  as  evidence  of  contamination  by 
sewage,  but  it  is  possible  for  the  bacillus  to  gain  access  to  the  water 
from  other  sources  also.  The  bacillus  is  present  in  the  excreta  of 
mammals  generally,  and  has  been  found  in  the  excreta  of  birds,  and 
in  surface  waters  there  will  undoubtedly  be  a  certain  amount  of 
contamination  caused  in  this  way.  The  question  as  to  whether  any 
contamination  of  this  kind  can  be  caused  by  various  fishes,  and  other 
forms  of  aquatic  life,  is  not  fully  established,  though  Eyre  has 
recently  found  the  B.  coli  in  the  excreta  of  fishes,  as  well  as  mammals 
and  birds.* 

*  Lancet,  1904,  i.,  p.  648. 


58  BACTERIA  IN  WATER 

Most  bacteriologists  would  condemn  a  water  containing  the 
typical  B.  coli  in  1  c.c.  as  showing  signs  of  sewage  pollution.  In  the 
case  of  a  recent  pollution  the  presence  of  B.  coli  affords  therefore  a 
much  more  delicate  test  of  pollution  than  any  chemical  examination 
which  can  be  made.* 

B.  enteritidis  sporogenes  is  another  organism  of  indication  as  to 
sewage  pollution,  and  its  presence  in  bacillary  form  or  as  spores  is 
now  accepted  as  showing  recent  or  remote  contamination. 

The  presence  of  streptococcus  is  held  by  many  bacteriologists  to 
be  a  sign  of  sewage  contamination,  although  some  contend  that  the 
presence  of  streptococci  does  not  indicate  dangerous  contamination 
unless  accompanied  by  B.  coli.  The  following  table  (p.  59),  from  the 
Thirty-fourth  Annual  Eeport  of  the  Lawrence  Sta.,  1903,  sets  forth, 
in  less  space  and  with  more  accuracy  than  could  be  recorded  in  many 
words,  the  relative  presence  of  the  chief  organisms  of  contamination, 
and  it  is  therefore  inserted. 

Lastly,  there  are  a  number  of  organisms  which  appear  to  be  fre- 
quently present  in  waters  contaminated  with  sewage,  and  are  rarely 
if  ever  found  in  pure  supplies.  The  occurrence  of  such  bacteria  in  a 
water  should  arouse  suspicion  as  to  its  origin  or  contamination. 
Among  this  group  of  bacteria  are  B.  fluorescens  putridus,  B.  erythro- 
spores,  B.  et  M.  urecey  B.  pyocyaneus,  B.  lactis  cyanogenus,  and  B. 
megaterium. 

(3)  The  presence  of  pathogenic  species. — The  presence  of  any 
pathogenic  organisms,  in  however  few  numbers,  is  of  course  sufficient 
for  the  condemnation  of  a  water.  For  instance,  the  presence  of  the 
bacillus  of  typhoid  fever  or  the  bacillus  of  cholera  at  once  condemns 
a  water.  There  are  very  few  authentic  records  of  such  organisms 
being  found,  and  it  is  therefore  necessary  to  judge  of  waters  by  the 
presence  of  organisms  of  contamination. 

Note. — A  water  may  be  considered  safe  and  potable  (a)  if  it 
contains  comparatively  few  organisms;  (b)  an  absence  of  organisms 
capable  of  fermenting  glucose  or  lactose  media;  (c)  an  absence  of 
B.  enteritidis  sporogenes;  and  (d)  an  absence  of  any  pathogenic 
species,  and  especially  if  these  conditions  are  found  to  exist  as  a 
result  of  several  examinations  or  of  periodic  examinations.  A  water 
should  be  condemned,  as  a  rule,  (a)  if  it  contains  a  very  large 
number  of  bacteria  per  c.c.  of  whatever  kind;  (b)  if  it  contains 
B.  coli  communis,  or  B.  enteritidis  sporogenes  or  streptococci  in  1  c.c.  or 
any  such  small  quantity;  (c)  if  it  gives  the  enteritidis  change  in  milk 
cultures ,  or  ferments  glucose  or  lactose  media.  It  should  be  con- 
demned without  hesitation  if  it  contains  B.  coli  and  B.  enteritidis  sporo- 
genes (or  spores),  and  streptococci,  or  if  it  contains  any  pathogenic 
organism,  in  however  small  a  quantity.  But  in  condemning  or 
*  See  also  Fourth  Report  Roy.  Com.  Sewage  Disposal,  1904,  pp.  106-109. 


ORGANISMS  OF  CONTAMINATION 


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60  BACTERIA  IN  WATER 

approving  a  water  supply  it  is  important  to  take  all  the  findings  of 
chemistry,  bacteriology,  and  topography  into  consideration.  The  whole 
history  of  the  sample  must  be  considered,  and  too  much  reliance 
must  not  be  placed  upon  the  mere  presence  or  absence  of  B.  coli,  or 
any  single  phenomenon  or  reaction.  No  ultimate  reliance  should,  as 
a  rule,  be  placed  upon  any  single  test. 

Natural  Purification  of  Water 

We  have  already  noticed  that  rivers  purify  themselves  as  they 
proceed.  There  are  many  excellent  examples  of  such  self- 
purification.  The  Seine  as  it  runs  through  Paris  becomes  highly 
polluted  with  every  sort  of  filthy  contamination.  It  receives  daily 
about  250,000  c.m.  of  sewage.  But  20  or  30  miles  below  the  city  it 
is  found  to  be  even  purer  than  above  the  city  before  it  received  the 
sewage.  In  small  rivers  it  is  the  same,  provided  the  pollution  is  less 
in  amount.  The  Thames  and  the  Severn  are  excellent  examples. 
Whilst  authorities  differ  with  regard  to  the  means  of  self-purification 
which  operate  most  effectually,  all  agree  that  in  some  way  rivers 
receiving  crude  sewage  are  able  in  a  marvellous  degree  to  become 
pure  again. 

The  chief  conditions  influencing  this  phenomenon  are  as  follow : — 

(a)  The  movement  of  the  water. — It  is  probable  that  any  beneficial 
result  accruing  from  this  cause  is  due  not  to  any  mechanical  factor  in 
the  movement,  but  to  the  extra  surface  of  water  available  for  oxida- 
tion processes.     Delepine  has  shown  that  the  effect  of  agitation  is  an 
increase  in  the  number  of  suspended  bacteria  which  he  attributes  to 
the  dislodgment  of  deposit  and  side  adhesions.     The  greatest  amount 
of  purification  in  his  experiments  occurred  when  the  rate  of  flow  was 
about  8  c.m.  per  hour.* 

(b)  The  pressure  of  the  water. — It  is  believed  that  the  volume  of 
water  pressing  down  upon  any  given  area  beneath  it  weakens  the 
vitality  of  certain  microbes.     In  support  of  this  theory,  it  is  urged 
that  the  number  of  bacteria  capable  of  developing  is  less  the  greater 
the  depth  from  the  surface.     Yet  it  must  be  remembered  that  mud  at 
the  bottom  of  a  river,  or  at  the  bottom  of  shallow  sea,  is  teeming  with 
living  organisms,  and  there  is  no  evidence  to  show  that  pressure  in 
river   water  ever  reaches  a  degree  capable  of  affecting  the  life  of 
bacteria.     Delepine  found  that  in  the  Manchester  mains  increase  of 
pressure  did  not  reduce  the  number  of  bacteria. f 

(c)  Light. — We  have  seen  how  prejudicial  is  light  to  the  growth 
of  organisms  in  culture  media.     This  is  so,  though  to  a  less  extent, 
in  water  (see  p.  18).     Arloing  held  that  sunlight  could  not  pierce 

*  The  Natural  Purification  of  Itunning  Water,  Jour,  of  Stale  Med.,  1901,  p.  517. 
f  Report  to  the  Manchester  Water  Works  Committee,  1894. 


NATURAL  PURIFICATION  OF  WATER  61 

a  layer  of  water  an  inch  in  thickness  and  still  act  inirnically  on 
micro-organisms.  But  Buchuer  found  that  the  sun's  rays  could 
pass  through  15  or  20  inches  and  yet  be  bactericidal.  This  evidence 
appears  contradictory.  On  the  whole,  however,  authorities  agree 
that  the  influence  of  the  sun's  rays  upon  water  is  in  some  degree 
bactericidal  and  causes  a  diminution  in  the  quantities  of  organisms 
after  acting  for  some  hours.  Especially  will  this  be  so  when  the 
water  is  spread  out  over  a  wide  area  and  is  therefore  shallow  and 
stationary,  or  moving  but  slowly.*  But  taken  as  a  whole  it  may 
be  said  that  light  does  not  exert  a  marked  influence  in  water  puri- 
fication. There  is,  on  the  other  hand,  evidence  to  prove  that  water 
in  its  passage  through  dark  mains  of  various  sizes  gradually  becomes 
deprived  of  a  great  part  of  its  bacterial  contents. 

(d)  Vegetation  in  water. — Pettenkofer,  in  his  observations  upon 
the  Iser  below  Munich,  has  shown  how  algse  bring  about  a  marked 
reduction    in   the   organic   matters   present  in   water.     Boyce    has 
pointed  out  that  in  the  river  Severn,  in  addition  to  the  temperature 
and  movement  being  unfavourable  to  B.  coli  and  presumably  patho- 
genic bacteria,  that  (a)  lack  of  pabulum,  and  (&)  antagonism  due  to 
the  fauna  and  flora  of  the  river  exert  an  unfavourable  influence  upon 
these   bacteria.     The   organic   matter  so  abundant  when  the  river 
becomes  polluted  at  Shrewsbury  is  diluted  and  destroyed  lower  down 
stream,  and  therefore  the  water  becomes  purified  of  bacteria  living 
on  the  organic  matter.     Fish,  birds,  rats,  protozoa,  and  forms  of  river 
life  generally  contribute  their  share  to  the  consumption  of  organic 
pabulum.     The   water  Ranunculus,  Spkcerotilus,   Lcptomitus  lactcus, 
sewage  fungi,  chlorophyll  containing  protophytes,  and  river  plants 
generally  assist  in  the  destruction  of  organic  matter  and  bacteria.f 

(e)  Dilution. — The  pollutions  passing  into  a  flowing  river  are  very 
soon  diluted  with  the  large  quantities  of  comparatively  pure  water 
always  forthcoming.     And  this,  whilst  it  lowers  the  percentage  of 
impurity,  also  raises  the  percentage  of  oxygenated  water.     Delepine 
has  pointed  out  as  a  result  of  artificial  experiments  that  dilution 
exerts  a  double  effect  on  the  bacterial  content  of  water.     In  the  first 
place  it  has  the  mechanical  effect  of  increasing  the  space  occupied  by 
a  definite  number  of  bacteria,  and  in  the  second  place  it  causes  a 
diminution  in  the  amount  of  pabulum  present  in  a  given  bulk  of  the 
impure  fluid.     Dilution  and  deposition  acting  together  exert  a  power- 
ful influence  as  purifiers.     Clark  and  Gage  of  the  Lawrence  station 
pointed  out  in  1903  that  the  number  of  B.  coli  in  a  polluted  river 
varies  in  inverse  ratio  with  the  dilution  of  the  entering  sewage  by 
the  river  water,  and  is  affected  by  the  temperature,  the  number  of 
B.  coli  being  larger  during  the  warm  weather  than  in  the  cold.     In 

*  See  also  Spitta's  work  on  the  Spree  at  Berlin,  Arcliw  fiir  Hyg.,  vol.  xxxviii. 
t  Roy.  Com.  on  Sewage  Disposal,  Second  Report,  1902,  pp.  104-109. 


62  BACTERIA  IN  WATER 

elHuents  from  water  filters  the  effect  upon  filter  ellbieney  of  dilution 
of  the  water  in  winter  is  less  marked  than  the  effect  of  high  tem- 
perature in  summer,  the  work  of  a  filter  in  warm  weather  being,  of 
course,  more  satisfactory  than  in  cold. 

(f)  Sedimentation. — Whilst  Pettenkofer  attributes  self-purifica- 
tion to  oxygenation  and  vegetation,  most  authorities  are  now  agreed 
that  it  is  largely  brought  about  by  the  subsidence  of  impure  matters, 
and  by  their  subsequent  disintegration  at  the  bottom  of  the  river. 
Sedimentation  and  side-adhesion  to  the  banks  in  rivers  and  streams 
of  solids  in  suspension  removes  a  large  number  of  bacteria  in  the 
Severn  (Boyce).  Sedimentation  obviously  is  greatest  in  still  waters. 
Hence  lake  water  contains  as  a  rule  very  few  bacteria.  "The 
improvement  in  water  during  subsidence  is  the  more  rapid  and  pro- 
nounced the  greater  the  amount  of  suspended  matter  initially 
present"  (Frankland).  Tils  has  pointed  out  that  the  number  of 
micro-organisms  was  invariably  smaller  in  the  water  collected  from 
the  reservoir  than  in  that  taken  from  the  source  supplying  the  latter. 
Percy  Frankland  has  demonstrated  the  same  effect  of  sedimentation 
by  storage  as  follows : — 

No.  of  Colonies  in 
1  c.c.  of  Water. 

1.  Intake  from  Thames,  25th  June  1892  .  1991 

2.  First  small  storage  reservoir           .  .  1703 

3.  Second  small  storage  reservoir      .  .  1156 

4.  Large  storage  reservoir      .            .  .  464 

The  large  reservoir  would  of  course  necessitate  a  prolonged  sub- 
sidence, and  hence  a  greater  diminution  than  in  the  small  reservoirs. 
Karlinski  gives  the  following  distribution  of  bacteria  in  the  Borka 
Lake  (Herzegovina) : — 

Bacteria  per  c.c. 
Surface  water   .  4000 


Five  inches  below  surface 

Ten  inches  below  surface 

Twelve  to  sixteen  inches  below  surface 

Bottom  when  mud  was  stirred  up 


1000 
600 
200 

6000 


Delepine  considers  that  bacteria  die  rapidly  in  the  deposit, 
although  their  large  numbers  are  evidence  of  the  effect  of  sedimenta- 
tion. He  examined  some  water  mains  after  the  sediment  had  been 
disturbed  and  also  with  the  sediment  undisturbed.  The  results 
were  as  follows  : — 

Sediment  undisturbed.  Sediment  disturbed. 

1.  51  living  bacteria  per  c.c.  334  living  bacteria  per  c.c. 

2.  356  ,,  „  3164 

3.  10  „  „  852 

He  concludes  (1)  that  sedimentation  is  a  very  important  factor 
of  bacterial  purification  in  flowing  water,  and  (2)  that  the  effects  of 


NATURAL  PURIFICATION  OF  WATER  63 

sedimentation  are  most  manifest  when  the  flow  of  water  is  rapid 
enough  to  prevent  the  accumulation  at  any  point  of  the  products  of 
bacterial  multiplication,  but  not  so  rapid  as  to  interfere  with  a 
comparatively  rapid  action  of  gravity.* 

In  the  case  of  a  tidal  stream  the  conditions  are  different,  as 
recently  pointed  out  by  Foulerton.j"  In  such  rivers  the  disease- 
producing  bacteria  are  deposited  not  only  on  the  bed  of  the  stream, 
but  also  on  the  mud,  or  sludge,  on  the  banks,  and  are  uncovered  by 
water  at  low  tide.  It  now  requires  only  the  agency  of  a  fly,  feeding 
first  on  the  organic  matter  in  the  sewage-contaminated  mud  and  then 
on  some  human  food,  milk  for  instance,  to  convey  the  bacillus  of 
typhoid  fever  from  the  river  to  some  human  being.  An  additional 
way  by  which  a  bacillus  of  this  kind  may  survive  after  it  has  been 
discharged  into  a  river  is  by  its  being  deposited  on  the  bed  of  the 
stream  where  there  are  shell-fish  layings.  It  has  been  proved  that 
the  typhoid  bacillus  can  survive  for  a  considerable  time  in  the  liquor 
contained  in  the  shell  of  the  oyster  or  the  mussel,  and  in  this  way  it 
may  escape  destruction  by  finding  itself  once  more  inside  the  con- 
sumer of  the  shell-fish.  Therefore,  in  the  case  of  sewage  discharged 
into  a  tidal  river,  owing  to  lack  of  dilution  and  sedimentation,  it  is  a 
menace  to  the  inhabitants  on  the  banks  in  one  or  both  of  these  ways. 
The  exact  degree  of  danger  depends  first  upon  the  extent  to  which 
the  sewage  is  purified  before  its  discharge  into  the  stream,  and 
secondly  upon  the  distance  from  the  source  of  pollution  at  which  con- 
tamination of  the  water  by  special  sewage  bacteria  is  still  appreciable. 

This  principle  of  sedimentation  operates  upon  all  bacteria,  which 
are  often  carried  down  on  gross  particulate  matter.  The  number 
of  B.  coli  is  reduced  quite  appreciably  by  storage  of  water  (Clark 
and  Gage).  Many  species  remain  in  the  mud,  sand,  or  other  deposit 
at  the  bottom  of  the  stream  or  reservoir.  The  parasitic  organisms 
die  on  account  of  the  unfavourable  environment. 

(g)  Oxidation. — Many  experiments  and  observations  have  been 
made  to  prove  that  large  quantities  of  oxygen  are  used  up  daily  in 
oxygenating  the  Thames  water.  Oxygenated  water  will  come  up 
with  the  tide  and  down  with  the  fresh  water  from  above  London. 
There  will  also  be  oxygen  absorption  going  on  upon  the  surface  of 
the  water,  and  from  these  three  sources  enough  oxygen  is  obtained 
to  oxidise  impurities  and  produce  what  is  really  an  "  effluent."  In 
many  smaller  streams  the  opportunity  for  oxidation  is  afforded  by 
weirs  and  falls. 

Probably  all  these  factors  play  a  part  in  the  self-purification  of 
rivers,  but  we  may  take  it  that  oxidation,  dilution,  and  sedimentation 
are  three  of  the  principal  agencies.  The  test  of  purification  is  in  the 

*  Jour.  ofStat.  Med.,  1901,  p.  518. 

t  Report  on  Pollution  of  Tidal  Ouse,  1903,  p.  11. 


64  BACTERIA  IN  WATER 

number  and  character  of  the  bacteria  at  different  stages  of  the 
river  (e.g.,  see  Table  of  Bacteria  in  Severn,  p.  38).  Jordan  has 
pointed  out  the  peculiar  value  of  the  reduction  of  B.  coli.* 

We  may  here  refer  in  passing  to  the  facts  obtainable  from  the 
late  Sir  Edward  Frankland's  report  on  Metropolitan  water  supply  in 
1894,  as  they  will  afford  a  connecting  link  between  natural  purifica- 
tion and  artificial  purification.  First,  judged  by  the  relatively  low 
proportion  of  carbon  to  nitrogen,  the  organic  matter  present  in  the 
water  was,  as  usual,  found  to  be  chiefly  of  vegetable  origin.  Secondly, 
an  immense  destruction  of  bacteria  was  effected  by  storage  in 
subsidence  reservoirs.  Thirdly,  the  bacterial  quality  of  the  water 
might  differ  widely  from  its  chemical  qualities.  It  is,  of  course,  a 
much  finer  index  of  pollution.  These  three  facts  are  of  primary 
importance  in  the  interpretation  of  water  reports,  and  it  will  be  well 
to  bear  them  in  mind.  Sir  E.  Frankland  also  referred  to  the  physical 
conditions  affecting  microbial  life  in  river  waters,  and,  as  in  previous 
reports,  to  the  importance  of  changes  of  temperature,  the  effect  of 
sunlight,  and  rate  of  flow.  Eespecting  the  relative  proportion  of 
these  factors,  he  wrote :  "  The  number  of  microbes  in  Thames  water  is 
determined  mainly  by  the  flow  of  the  river,  or,  in  other  words,  by 
the  rainfall,  and  but  slightly,  if  at  all,  by  either  the  presence  or 
absence  of  sunshine,  or  a  high  or  low  temperature.  With  regard  to 
the  effect  of  sunshine,  the  interesting  researches  of  Dr  Marshall 
Ward  leave  no  doubt  that  this  agent  is  a  powerful  germicide,  but  it 
is  probable  that  the  germicidal  effect  is  greatly  diminished,  if  not 
entirely  prevented,  when  the  solar  rays  have  to  pass  through  even 
a  comparatively  thin  stratum  of  water  before  they  reach  the  living 
organisms."  Subsequent  investigations  have  confirmed  the  im- 
portance of  these  broad  principles,  and  from  which  it  is  clear  that 
evidence  favours  the  effect  of  sedimentation  and  dilution.  These  two 
factors  in  conjunction  with  filtration  are,  practically  speaking,  the 
methods  of  artificial  water  purification,  to  which  reference  will  now 
be  made. 

Artificial  Purification  of  Water 

Sedimentation  and  Precipitation. — In  nature  we  see  this 
factor  in  operation  in  lakes  and  reservoirs.  For  example,  the  water 
supply  of  Glasgow  is  the  untreated  overflow  from  Loch  Katrine. 
Purification  has  been  brought  about  by  means  of  subsidence  of 
impurities.  Nothing  further  is  needed.  Much  of  the  purification 
obtained  in  reservoirs  supplying  large  towns  is  due  to  the  same 
factor.  Artificially  we  find  it  is  this  factor  which  is  the  mechanical 
purifier  of  biological  impurity  in  such  methods  as  Clark's  process. 
By  this  mode  "temporary  hardness,"  or  that  due  to  soluble 

*  Jour.  ofHyg.,  1901,  p.  293. 


DUST  AND  AIR  POLLUTION  81 

suitable  nidus  for  putrefactive  bacteria.  The  large  quantities  of 
bacteria  which  a  decayed  tooth  contains  are  easily  demonstrated. 

From  the  two  series  of  experiments  which  we  have  now  con- 
sidered we  may  gather  the  following  facts : — 

(a)  That  air  may  contain  great  numbers  of  bacteria  which  may 
be  readily  inspired. 

(&)  That  in  health  those  inspired  do  not,  as  a  rule,  pass  beyond  the 
moist  surface  of  the  nasal  and  buccal  cavities,  except  in  persons  who 
practice  oral  instead  of  nasal  respiration. 

(c)  That  in  the  nose  and  mouth  there  are  various  influences  of  a 
bactericidal  nature  at  work  in  defence  of  the  individual. 

(d)  That  expired  air  in  normal  quiet  breathing  contains,  as  a 
rule,  no  bacteria  whatever. 

The  practical  application  of  these  things  is  a  simple  one.  To, 
keep  air  free  from  bacteria,  the  surroundings  must  be  moist.  Strong 
acids  and  disinfectants  are  not  required.  Moisture  alone  will  be 
effectual.  Two  or  three  examples  at  once  occur  to  the  mind. 
Anthrax  spores  are  conveyed  from  time  to  time  from  dried  infected 
hides  and  skins  to  the  hands  or  bodies  of  workers  in  warehouses  in 
Bradford,  Bermondsey,  Finsbury,  and  other  places.  If  the  surround- 
ings are  moist  and  the  hides  moist,  anthrax  spores  and  other  bacteria 
do  not  remain  free  in  the  air.  As  a  matter  of  actual  experience,  it  has 
been  found  that  handling  dried  hair  or  dried  skins  leads  to  more  anthrax 
infection  than  handling  the  same  articles  in  a  moist  condition.* 

Again,  the  bacilli  (or  "  spores  ")  of  tuberculosis  present  in  sputum 
in  great  abundance  cannot  infect  the  air  until  and  unless  the 
sputum  dries.  So  long  as  the  expectorated  matter  remains  on  the 
pavement  or  handkerchief  wet,  the  surrounding  air  will  derive  from 
it  no  bacilli  of  tubercle.  But  when  in  the  course  of  time  the  sputum 
dries,  then  the  least  current  of  air  will  at  once  infect  itself  with  the 
dried  spores  or  bacilli.  It  should,  however,  be  remembered  that  the 
"  cough-spray  "  and  microscopic  particles  of  saliva  emitted  in  shout- 
ing, heavy  breathing  through  the  mouth,  etc.,  have  been  shown  by 
Fliigge  and  others  to  carry  the  bacilli  of  tubercle.  Such  conveyance 
may,  of  course,  prove  a  channel  of  infection  between  diseased  and 
healthy  persons.  The  typhoid  bacillus,  too,  occupies  the  same  position. 
Only  when  the  excrement  dries  can  the  contained  bacteria  infect 
the  air.  It  is  of  course  well  known  that  the  common  channel  of 
infection  in  typhoid  fever  is,  not  the  air,  whereas  the  reverse  holds 
true  of  tuberculosis.  But  if  it  happens  that  the  excrement  of 
patients  suffering  from  typhoid  dries,  the  air  may  become  infected ; 
if,  on  the  other  hand,  it  passes  in  a  moist  state  into  the  sewer,  even 
though  untreated  with  disinfectants,  all  will  be  well  as  regards  the 
surrounding  air. 

*  Annual  Reports  of  Medical  Inspector  of  Factories  and  Workshops,  1902  and  1903. 

F 


82  BACTERIA  IN  THE  AIR 

A  still  more  remarkable  illustration  of  the  effect  of  a  moist 
perimeter  upon  the  contained  bacteria  is  to  be  found  in  sewer  air. 
For  long  it  has  been  known  that  air  polluted  by  sewage  emanations 
is  capable  of  giving  rise  to  various  degrees  of  ill-health.  These 
chiefly  affect  two  parts  of  the  body;  one  is  the  throat  and  the  other 
the  intestine.  Irritation  and  inflammation  may  be  set  up  in  both  or 
either  by  sewer  air.  Such  conditions  are  in  all  probability  produced 
by  a  lowering  of  the  resistance  and  vitality  of  the  tissues,  and  not  by 
a  conveyance  of  bacteria  in  sewer  air  or  by  any  stimulating  effect 
upon  bacteria  exercised  by  sewer  air.  What  evidence  we  have  is 
against  such  factors.  Several  series  of  investigations  have  been 
made  into  the  bacteriology  of  sewer  air,  amongst  others  by  Uffel- 
mann,  Carnelly  and  Haldane,  and  Laws  and  Andrewes.  From  their 
Jabours  we  may  formulate  four  simple  conclusions : — 

1.  The  air  of  sewers  contains  very  few  micro-organisms  indeed, 
sometimes  not  more  than  two  organisms  per  litre  (Haldane),  and 
generally  fewer  than  the  outside  air  (Laws  and  Andrewes). 

2.  There  is   not,  as  a   rule,  intimate   relationship  between   the 
microbes  contained  in  sewer  air  and   those   contained  in   sewage. 
Indeed,  there  is  a   marked   difference  which   forms   a   contrast   as 
striking  as  it  is  at  first  sight  unexpected.    The  organisms  isolated  from 
sewer  air  are  those  commonly  present  in  the  open  air.     Micrococci 
and  moulds  predominate,  whereas  in  sewage  moulds  and  micrococci 
are  rare,  and  bacilli  are  most  numerous.     Liquefying  bacteria,  too, 
which   are   common   in   sewage,  are   extremely  rare   in   sewer   air. 
Bacillus   coli  communis,   which   occurs   in   sewage   from   20,000    to 
200,000  per  c.c.,  is  altogether  absent  from  sewer  air. 

3.  As  a  rule  it  may  be  said  that  only  when  there  is  splashing  in 
the  sewage,  or  when  bubbles  are  bursting  (Carnelly  and  Haldane),  is 
it  possible  for  sewage  to  part  with  its  contained  bacteria  to  the  air 
of   the   sewer.      But  under   these  conditions   it  may  part  with   a 
considerable  number. 

4.  Pathogenic  organisms  and  those   nearly  allied  to   them   are 
found  in  sewage,  but  are  absent  in  sewer  air.     Uffelmann  isolated 
the  Staphylococcus  pyogenes  aureus   (one   of   the  organisms  of  sup- 
puration), but  such  a  species  is  exceptional  in  sewer  air.     Hence, 
though  sewer  air  is  popularly  held  responsible  for  directly  conveying 
virulent  micro-organisms  of   various   diseases,   there   is   up   to   the 
present  no  evidence  of  a  substantial  nature  in  support  of  such  views. 
In  1894,  Laws  and  Andrewes  found  an  average  of  2,781,650  bacteria 
per  c.c.  in  fresh  sewage,  and  in  older  sewage  from  3,400,000  per  c.c., 
to  11,216,000,  and  they  pointed  out  that  temperature  and  dilution 
of  sewage  were  determining  factors  in  the  number  of  bacteria  present. 
They  consider  that  sewage  may  become  a  medium  for  the  dissemina- 
tion of  the  typhoid  bacillus,  and  that  sewage-polluted  soil  may  possibly 


BACTERIA  IN  SEWER  AIR  83 

give  up  germs  to  the  subsoil  air,  but  they  are  satisfied  that  the  air 
of  sewers  themselves  does  not  play  any  part  in  the  conveyance  of 
the  typhoid  bacillus.* 

In  passing,  mention  may  be  made  of  some  interesting  observations 
recorded  by  Mr  S.  G.  Shattock  on  the  effect  of  sewer  air  upon  the 
toxicity  of  lowly  virulent  bacilli  of  diphtheria.  Some  direct  relation- 
ship, it  has  been  surmised,  exists  between  breathing  sewer  air  and 
"catching"  diphtheria.  Clearly,  it  cannot  be  that  the  sewer  air 
contains  the  bacillus.  But  some  have  supposed  that  the  sewer  air 
has  had  a  detrimental  effect  by  increasing  the  virulent  properties  of 
bacilli  already  in  the  human  tissues.  Two  cultivations  of  lowly 
virulent  B.  diphtheria  were  therefore  grown  by  Mr  Shattock  in  flasks 
upon  a  favourable  medium  over  which  was  drawn  sewer  air.  This 
was  continued  for  two  months  in  the  one  case,  and  five  weeks  in  the 
other.  Yet  no  increased  virulence  was  secured.f  Such  experiments 
require  ample  confirmation,  but  even  now  it  may  be  said  that  sewer 
air  does  not  necessarily  have  a  favouring  influence  upon  the  virulence 
of  the  bacilli  of  diphtheria.  Such  experiments  do  not  affect  the 
contrary  question  of  the  possibility  of  sewer  air  depressing  the  vitality 
of  the  individual,  and  so  allowing  even  lowly  virulent  bacilli  to  do 
mischief.  Of  such  depression  caused  by  breathing  sewer  air  there 
is  clinical  proof,  and  although  sewer-men  do  not  appear  to  be  affected, 
persons  freshly  breathing  sewer  air  may  be. 

It  should  be  noted  that  the  bacilli  of  diphtheria  are  capable  of 
lengthened  survival  outside  the  body,  and  are  readily  disseminated 
by  very  feeble  air  -  currents.  The  condition  necessary  for  their 
existence  outside  the  body  for  any  period  above  two  or  three  days 
is  moisture.  Dried  diptheria  bacilli  soon  lose  their  vitality.  It  is 
possible,  owing  to  this  fact,  that  the  disease  is  not  as  commonly  con- 
veyed by  air  as,  for  example,  tubercle. 

3.  The  Influence  of  Gravity  upon  bacteria  in  the  air  may  be 
observed  in  various  ways,  in  addition  to  its  action  within  a  limited 
area  like  a  sewer  or  a  room.  Miquel  found  in  some  investigations 
in  Paris  that,  whereas  on  the  Eue  de  Eivoli  750  germs  were  present 
in  a  cubic  metre,  yet  at  the  summit  of  the  Pantheon  only  28  were 
found  in  the  same  quantity  of  air.  Frankland  found  that  air  at  the 
top  of  Primrose  Hill  contained  9  organisms  per  ten  litres,  and  air 
at  the  bottom  24.  On  the  spire  of  Norwich  Cathedral  (310  feet), 
ten  litres  of  air  yielded  7  organisms,  on  the  tower  (180  feet)  9,  and 
on  the  ground  18.  At  the  level  of  the  golden  gallery  of  St  Paul's 
Cathedral  he  found  in  every  ten  litres  11  bacteria,  at  the  stone  gallery 
34,  and  in  St  Paul's  Churchyard  70.  As  Tyndall  has  pointed  out, 

*  Report  to  the  London  County  Council  on  the  Result  of  Investigation  on  the  Micro- 
organisms of  Salvage,  by  J.  Parry  Laws  and  F.  W.  Andrewes,  1894,  p.  14. 
f  Pathological  Society  of  London,  Transactions,  1897. 


84 


BACTERIA  IN  THE  AIR 


even  ultra-microscopic  cells  obey  the  law  of  gravitation.  This  is  equally 
true  in  the  limited  areas  of  a  laboratory  or  warehouse,  and  in  the  open 
air.  At  high  altitudes,  the  air  may  be  looked  upon  as  practically 
germ-free,  although  here  again  the  lighter  spores  of  the  mould  fungi 
may  cause  them  to  be  carried  by  air  currents  to  a  very  great  height. 
In  the  recent  researches  of  Dr  Jean  Binot  of  the  Pasteur  Institute,* 
100  litres  of  air  taken  at  the  summit  of  Mont  Blanc  did  not  contain 
a  single,  microbe,  and  the  total  number  of  organisms  varied  between 
4  and  11  per  metre  cube  (1000  litres).  An  examination  of  the  air  of 
the  interior  of  M.  Janssen's  Observatory,  situated  on  the  highest 
point  of  Mont  Blanc,  and  taken  in  two  different  rooms,  gave,  on  the 
other  hand,  540  and  260  organisms  per  metre  cube.  The  gradual 
increase  of  the  number  of  organisms  as  descent  to  lower  level  takes 
place  is  of  interest.  Thus  6  per  metre  cube  were  found  in  the  Grand 
Plateau,  8  at  the  Grand  Mulet,  and  14  at  the  Plon  de  1* Aiguille. 
Upon  the  Mer  de  Glace  23  organisms  were  found,  and  49  at  Montan- 
vert.  Graham  Smith  found  that  at  the  top  of  the  Clock  Tower  of 
the  Houses  of  Parliament  in  London  there  was  only  about  one-third 
of  the  number  of  bacteria  found  at  the  ground  level. f 

4.  Air  Currents. — Miquel,  Pasteur,  Cornet,  and  other  workers 
have  shown  that  the  presence  of  micro-organisms  in  air  depends  in 
part  upon  air  currents,  winds,  etc.  In  the  month  of  August,  with 
the  wind  from  the  south,  i.e.  blowing  from  the  country  citywards,  the 
number  of  organisms  was  found  by  Miquel  to  be  40  in  the  Mont  Souris 
Pare  around  the  Observatory,  while  at  the  same  moment  a  record  of 
14,800  was  obtained  in  the  4th  Arrondissement,  which  may  be  taken 
as  the  centre  of  Paris,  and  comprises  the  surroundings  of  Notre  Dame 
and  of  the  Hotel  de  Ville.  In  the  month  of  June,  on  the  other  hand, 
with  the  wind  blowing  from  the  N.E.,  i.e.  across  the  city  towards 
Mont  Souris,  the  numbers  were,  in  the  4th  Arrondissement,  10,000  per 
metre  cube,  and  in  the  Park  of  Mont  Souris  itself,  1180  per  metre  cube. 

The  seasonal  variations  of  the  organisms  present  in  the  air  are 
also  worthy  of  note,  and  depend  chiefly  upon  dust  and  air  currents. 
The  following  table  shows  the  mean  over  a  period  of  ten  years  in  the 
air  taken  at  Mont  Souris  : — 


Average  per  metre  cube. 

Season. 

Bacteria. 

Moulds. 

Winter  . 
Spring  . 
Summer 
Autumn 

170 
327 
480 
195 

175 
145 
210 
235 

*  Communication  a  VAcad6mie  des  Sciences  de  Paris,  17  Mars  1902. 
f  Jour,  of  Hyg.,  1903,  p.  513. 


INFLUENCE  OF  CARBONIC  ACID  GAS  85 

Similar  experiments  have  been  carried  out  by  Frankland,  Fliigge, 
Delalivesse,  Neisser,  Chick,  Andrewes,  and  others.*  The  last  named 
conducted  some  experiments  in  London  streets  in  1902,  and  reported 
his  results  to  the  Pathological  Society.  He  found  the  number  of 
organisms  varied  greatly,  but  no  pathogenic  species  were  detected. 
The  four  species  he  isolated  were  staphylococci,  sarcinse,  strepto- 
thricesB,  and  moulds. 

Carnelly,  Haldane,  and  Anderson  found  the  ratio  of  organisms 
in  the  air  increased  according  to  whether  the  air  was  examined  on 
still  damp  days,  windy  damp  days,  still  dry  days,  and  windy  dry 
days,  and  in  brief  this  expresses  the  findings  of  most  investigators. 

Some  new  light  has  been  thrown  upon  the  subject  of  pathogenic 
organisms  in  air  by  Neisser  in  his  investigations  concerning  the 
amount  and  rate  of  air  currents  necessary  to  convey  certain  species 
through  the  atmosphere.  He  states  that  the  bacteria  causing 
diphtheria,  typhoid  fever,  plague,  cholera,  and  pneumonia,  and 
possibly  the  common  Streptococcus  pyogenes,  are  incapable  of  being 
carried  by  the  molecules  of  atmospheric  dust  which  the  ordinary 
insensible  currents  of  air  can  support,  whilst  Bacillus  anthracis,  B. 
pyocyaneus,  and  the  bacillus  of  tubercle  are  capable  of  being  aerially 
conveyed.  This  work  will  require  further  confirmation  before  being 
entirely  accepted. 

Finally,  some  mention  may  be  made  of  the  relationship  alleged 
to  exist  between  the  presence  of  a  considerable  degree  of  carbonic 
acid  gas  in  an  atmosphere  and  the  number  of  bacteria  contained  in 
the  same  atmosphere.  As  far  as  may  be  judged,  it  would  appear 
that  the  relationship  is  but  slight.  But  to  illustrate  the  subject  as 
well  as  other  points  of  importance  in  the  bacteriology  of  air,  four 
separate  investigations  may  be  mentioned. 

(i.)  Haldane  and  Osborn,  in  their  inquiry  into  the  ventilation  of 
factories  and  workshops,  made  a  number  of  bacteriological  examina- 
tions.f  The  determinations  of  bacteria  were  made  by  a  slightly  modified 
form  of  Frankland's  method.  The  air  was  drawn  through  a  sterilised 
plug  of  glass  wool  by  means  of  a  brass  syringe  of  known  capacity. 
The  glass  tubes  containing  the  glass  wool  plugs  were  each  enclosed 
in  a  separate  outside  sterilised  glass  tube,  with  an  asbestos  plug. 
In  taking  the  sample  of  air  the  inside  tube  was  attached  directly  to 
the  pump  by  means  of  a  short  piece  of  stout  rubber  tubing.  The 
plug  was  afterwards  transferred  with  the  necessary  precautions  to  a 
shallow,  flat-bottomed  flask,  containing  a  small  quantity  of  liquefied 
gelatine,  which  was  shaken  so  as  to  disintegrate  and  spread  the  glass 

*  See  also  Jour,  of  Sanitary  Institute  (Oct.  1902),  vol.  xxiii.,  pt.  iii.,  p.  209-236. 
The  Dust  Problem,  by  Sir  J.  Crichton-Browne,  F.R.S. 

t  First  Report  of  the  Departmental  Committee  appointed  to  inquire  into  the 
Ventilation  of  Factories  and  Workshops,  1902.  J.  S.  Haldane,  M.D.,  F.R.S.,  and 
E.  H.  Osborn. 


86 


BACTERIA  IN  THE  AIR 


wool.  The  gelatine  having  set,  the  flask  was  incubated  at  20  C.  till  no 
further  colonies  of  bacteria  or  moulds  developed.  Some  of  the  chief 
results  were  as  follows : — 


Cub. 
Content. 

CO2 

per  10,000"  parts. 

Bacteria  and  Moulds 
per  Litre  of  Air. 

Inside. 

Outside  Air. 

Bacteria. 

Moulds. 

Tailor,  Whitechapel.        .     .  ., 

67,500 

35'8 

3-5 

17 

22 

•>1  953 

9  '2 

3  '5 

8 

1 

•>  750 

4'6 

3-5 

16 

2 

IS  636 

lO'O 

3'5 

9 

8 

9  800 

7'4 

3-5 

9 

0 

London,  E.    .        ... 

27,265 

14-6 

3-5 

10 

2 

London,  E.G. 

26,460 

14-6 

3-5 

25 

3 

Capmaker,  London,  E.     . 

4,296 

23-0 

3-5 

9 

2 

Dressmaker,  London,  W. 

21,600 

13-2 

3-5 

8 

0 

Boot  Workshop,  London,  E.    . 

8,688 

8-8 

3-5 

25 

6 

Railway  Works,  Wilts.     . 

93,786 

4-6 

3-5 

20 

2 

Chocolate  Factory,  Bermondsey 
Newspaper  Printer,  Lond.  E.  C. 

12,000 
24,098 

6'2 
16-5 

3'5 
3-5 

8 
9 

0 
0 

»              »»                   » 

45,259 

15-2 

3'5 

6 

6 

»»              »»                   » 

23,562 

25'4 

3-5 

10 

2 

Ropemaker,  Chatham  *             . 

... 

20 

6 

»»                  »»                      • 

82 

8 

»»                  »»                      • 

... 

850 

18 

*  The  ventilation  of  this  large  room  was  considerable,  but  having  the  effect  of  keeping  dust  in 
suspension  rather  than  expelling  it  from  the  room.  Three  tests  made  here  were  all  in  the  same  work- 
place,  differing  only  in  degree  of  dust  present. 

(ii.)  In  1902  the  writer  made  some  observations  in  Finsbury  on  the 
number  of  bacteria  to  be  found  in  the  air  of  underground  bakehouses. 
Four  were  selected,  and  the  degree  of  carbonic  acid  gas  was  estimated 
by  Pettenkofer's  method,  and  examinations  were  made  as  follow  of 
the  bacteria  pollution.  In  each  of  these  bakehouses,  whilst  work 
was  going  on,  three  agar-plates  (of  9 '6  inches  area  each)  were  exposed 
for  thirty  minutes.  One  plate  was  placed  on  the  floor,  one  on  the 
table  or  trough  where  the  bread  was  being  made,  and  one  on  a  shelf 
near  the  ceiling.  After  exposure  for  thirty  minutes  the  plates  were 
re-covered  and  incubated  at  blood-heat  (37°  C.),  for  exactly  twejity- 
two  hours.  All  the  plates  then  showed  abundant  growth.  Doubtless 
if  the  plates  had  been  incubated  for  forty-eight  hours,  or  three  or 
four  days,  there  would  have  been  a  greater  growth  of  colonies,  and 
it  is  probable  also  that  if  some  of  the  plates  had  been  placed  at  room 
temperature  certain  bacteria  would  have  grown  which  did  not  appear 
at  blood-heat  in  twenty -two  hours.  It  is  not  suggested  that  these 
plates  provide  an  adequate  record  of  the  bacteria  present  in  the  air 
of  these  bakehouses.  The  object  was  merely  to  obtain  a  comparative 


f>LATE  7. 
AIR-PLATES  EXPOSED   IN   BAKEHOUSES  (30  minutes). 


(i.)  Above-ground  Bakehouse  (Z.)    Agar  culture,  22  hours  at  37°  C. 


(ii.)  Under-ground  Bakehouse  (C.)    Agar  culture,  22  hours  at  37°  C 

[To  face  page  86. 


BACTERIA  IN  BAKEHOUSE  AIR 


87 


idea  of  the  air  of  underground  bakehouses  and  above-ground  bake- 
houses in  Finsbury.  Accordingly,  the  whole  of  the  30  plates  used 
in  this  examination  were  treated  exactly  the  same  in  every  way,  the 
medium,  exposure,  and  temperature  and  period  of  incubation  being 
precisely  similar.  The  results,  therefore,  whilst  of  little  value  as  a 
complete  examination  of  the  air,  are  useful  and  reliable  for  comparison 
with  each  other. 

The  results  were  as  follow : — 


Carbonic  Acid  Gas 

Average  No.  of 

in  parts 

Bacteria 

per  10,000  (Col  well). 

on  each  Plate. 

Underground  Bakehouse 

B 

12'0 

800 

»»                   >» 

C 

17-5 

680 

»»                   »» 

D 

16-9 

600 

E 

13'6 

600 

Above-ground  Bakehouse 
Outside  Air  in  street 

Z 

(C) 

4-9 
4-5 

200 
160 

Inside  the  bakehouses  there  was  also  an  interesting  distribution 
of  bacteria  as  follows : — 


No.  of  Bacteria 
per  Plate  on 
Shelf. 

No.  of  Bacteria 
per  Plate  on 
Table. 

No.  of  Bacteria 
per  Plate  on 
Floor. 

Underground  Bakehouse     C 
Above-ground  Shop  of         C 
Above-ground  Bakehouse    Z 

490 
130 
150 

720* 
150 
170* 

850 
720 
300 

Illustrations  of  these  two  plates  are  attached  (Plate  7). 


From  these  figures  it  will  be  seen  (a)  that  underground  bakehouse 
air  contained  at  least  four  times  more  bacteria  than  street  air  around 
it ;  (5)  at  least  three  times  more  bacteria  than  the  air  of  the  shop 
over  it ;  and  (c)  at  least  three  times  more  bacteria  than  the  above- 
ground  bakehouse.  The  general  result  of  the  investigations  was  that 
the  air  of  the  typical  underground  bakehouses  examined  (1)  contained 
14*8  volumes  per  10,000  of  carbonic  acid  gas,  C02  (as  compared  with 
4*9  in  above-ground  bakehouses  and  4*3  in  the  streets  of  Finsbury) ; 
(2)  that  it  contained  between  10  and  24  per  cent,  less  moisture  than 
outside  air  surrounding  the  bakehouses ;  and  (3)  that  it  contained  at 
least  four  times  more  bacteria  than  surrounding  street  air,  and  three 


88 


BACTERIA  IN  THE  AIR 


times  more  bacteria  than  the  air  of  a  typical  above-ground   bake- 
house.* 

Dr  Scott  Tebb  has  also  made  a  somewhat  parallel  examination 
of  the  air  of  London  streets  as  compared  with  the  railway  tube  of 
the  City  and  South  London  railway.f  As  the  result  of  a  large 
number  of  investigations  carried  out  in  a  similar  way  to  the  writer's 
examinations  in  bakehouses,  the  following  figures  were  arrived  at : — 


No.  of 

Air  of 

UU2 

per  10,000  parts. 

Micro-organisms 
per  Plate. 

The  open  streets       .... 

3'8 

459 

Platforms  in  Railway  Tube      . 
Railway  Carriages  in  Tube 

7'9 
.    11-6 

114 
218 

(iii.)  Thirdly,  some  of  the  results  of  the  investigations  of  Graham 
Smith  into  the  condition  of  the  atmosphere  of  the  House  of  Commons 
may  be  mentioned.^  He  used  a  modification  of  Frankland's  method  of 
filtering  the  air  to  be  examined  (4'5  litres  in  each  case)  through  glass 
wool.  An  air-pump  and  a  rubber  tube  of  10  feet  in  length  were 
used  for  drawing  the  air  through,  and  gelatine  was  used  as  the 
medium,  the  cultures  being  incubated  at  20°  C.  for  five  days  or 
longer.  The  results  may  be  expressed  in  tabular  form  in  three 
series : — 


Experiments  on  Outside  Air,  1 

8th  July. 

No.  of 

No.  of  Moulds 

Position. 

Bacteria  and  Moulds 

only 

per  litre. 

per  litre. 

1.  Terrace  (ground  level)    . 
2.         ,,       (10  feet  from  ground) 

4'2 
2'9 

I'l 
1-1 

3.         „       (20  feet  from  ground) 

3'3 

2'0 

4.  Clock  Tower  (half-way  up)     . 
5.            „            (top)  . 
6.  Peers'  Inner  Court 

1-5 
1-3 
4-2 

0-2 
0-6 
0'6 

7.  Star  Court       

6-0 

1'3 

Similar  experiments  were  performed  in  the  House  itself  during  a 

*  Report  on  Bakehouses  in  Finsbury  (Newman),  1902,  p.  51. 

t  Report  of  Public  Analyst  of  Southwark  on  Condition  of  Air  on  City  and  South 
London  Railway,  1903.     W.  Scott  Tebb,  M.D. 
J  Jour.  o/Hyg.,  1903,  pp.  498-513. 


OF  HOUSE  OF  COMMONS 


89 


sitting.     The  air  as  it  entered  the  House  contained  2 '6  bacteria  and 
moulds  per  litre. 


Experiments  in  Debating  Chamber,  21st  July.     Bacteria  and  Moulds  per  litre. 

Position  of  Examination. 

Early 
Series. 

Early 
Series. 

Late 

Series. 

Late 
Series. 

Average 
of  all 
Experiments. 

7   P.M. 

7.15  P.M. 

10.30  P.M. 

10.45  P.M. 

1.  Government  Side  (third  seat)  . 

10-6 

5-2 

7-0 

6'0 

7-2 

2.             „              „    (back  seat)  . 
3.  Opposition  Side  (third  seat)    . 

5-1 

4-4 
6-2 

4-4 
5'2 

5*2 

5-7 

4-8 
5'4 

4.  Equalising  Chamber 

8-0 

9'2 

8-4 

7-7 

8-3 

(Air  before  it  passes  into  Debating 

Chamber.) 

5.  Roof        

8-8 

8'2 

7-0 

6-4 

7'6 

A  third  series  of  examinations  was  made  by  Graham  Smith  of 
the  air  in  certain  committee  rooms,  etc.,  as  follows : — 


Experiments  in  Committee,  Dining,  and  Smoking  Booms. 

No.  of 

No.  of  Moulds 

Position  of  Examination. 

Bacteria  and  Moulds 

only 

per  litre. 

per  litre. 

1.  Committee    Room    9,    fans    working,     150 

persons  present,  1.45  P.M.  .... 

13-3 

4-0 

2.  Committee    Room     9,     fans     working,    150 

persons  present,  1.45  P.M.  . 

20-9 

4-6 

3.  Committee   Room   1,   fans   not  working,  41 

persons  present,  1.30  P.M  

35-5 

5'3 

4.  Committee   Room  1,  fans   not  working,  41 

persons  present,  1.30  P.M.  .... 
5.  Central    Dining-room,   36    persons    present, 

33'7 

4-2 

8  P.M.                

41-3 

8-4 

6.  Central    Dining-room,   36    persons    present, 

8  P.M.                

44-2 

12'0 

7.  Members'  Smoking-room,  24  persons  present, 

9  P.M  

30-6 

10-6 

8.  Members'  Smoking-room,  24  persons  present, 

9  P.M.                

8-6 

4-4 

Separate  investigation  as  to  the  degree  of  C02  present  in  the 
Debating  Chamber  of  the  House  of  Commons  revealed  between  3-4 
parts  per  10,000. 

Dr  Graham  Smith,  as  a  result  of  his  investigations,  arrived  at 
the  following  conclusions : — 

1.  The  number  of  micro-organisms  in  the  open  space  surrounding 
the  House  of  Parliament  is  comparatively  small  (4*2  per  litre). 


90  BACTERIA  IN  THE  AIR 

2.  The  air  in   the   debating  chamber  is  from  a   bacteriological 
point  of  view  remarkably  pure  (5*8  per  litre  as  average  of  eleven 
experiments). 

3.  The  number  of  bacteria  found  in  the  committee,  dining,  and 
smoking  rooms  was  several  times  greater  than  in  the  chamber  (32 '3 
per  litre  as  average  of  six  experiments). 

4.  No  organisms  pathogenic  to  man  were  isolated,  and  only  a  few 
which  were  pathogenic  to  animals. 

(iv.)  Fourthly,  in  1902  Andre wes  furnished  a  report  to  the  London 
County  Council  on  the  micro-organisms  present  in  the  air  of  the  tube 
of  the  Central  London  Kail  way.*  The  method  he  employed  was 
in  principle  that  of  Frankland,  viz.,  the  aspiration  by  means  of  a  brass 
syringe  (capacity  425  c.c.)  of  a  known  volume  of  air  (5  litres),  through 
a  plug  of  glass  wool  and  finely-powdered  cane-sugar.  The  latter 
retains  the  micro-organisms,  which  can  be  subsequently  distributed 
through  a  suitable  cultivating  medium  (such  as  gelatine)  in  a  Petri 
dish.  The  gelatine  plate-cultures  were  incubated  at  20°  C.  for  four 
days,  when  the  colonies  were  counted,  examined,  and  sub-cultured. 
Special  control  experiments  were  made,  and  search  was  also  made 
for  the  presence  of  anaerobic  organisms.  The  twelve  series  of 
experiments  yielded  results  which  may  be  abstracted  and  tabulated 
as  follow : — 


a    ^ 

"a 

i 

s-o2 

4 

01 

t>> 

^ 

i 

j§  ^ 

m 

•s 

1? 

a 

O 

si* 

1 

•a 

O 

g 

Q 

g 

•I 

1*1 

'o 

* 

H 

d 

1 

d' 
o 

S°o" 
^o'oS 

d 

O 

1 

i    j? 

to 

O 

1.  Platform     . 

11.30a.m. 

66 

30-3 

•09 

•0019 

14 

13 

2.  Lift     . 

11.45  a.m. 

61 

30'5 

•109 

•0028 

20 

19 

3.  Carriage  (smoking) 
4.  Tunnel 

11.45a.m. 
11.45  a.m. 

70 

67 

29-5 
30-2 

•108 
•082 

•0026 
•0010 

51 

10 

10 

8 

5.  Carriage  (non-smoking) 
6.  Platform      . 

2.45  p.m. 
5.10  p.m. 

68 
67 

30-5 
30-1 

•111 
•103 

•0027 
•0012 

13 
30 

14 
11 

7.  Platform      . 

11.40  a.m. 

68 

30-4 

•094 

•0010 

106 

6 

8.  Carriage  (non-smoking) 
9.  Tunnel 

11.20  a.m. 
11.15a.m. 

72 
70 

30-2 
30-2 

•134 
•104 

•0016 
•0018 

90 
10 

13 

4 

10.  Lift     .... 

11.0    a.m. 

66 

29-9 

•152 

•0042 

64 

9 

11.  Staircase,  Passage 

11.0    a.ra. 

64 

29-8 

•078 

•0013 

13 

3 

12.  Staircase,  Passage 

11.0    a.m. 

68 

30-0 

•102 

•0023 

17 

7 

*  Examination  of  the  Atmosphere  of  the  Central  London  Railway,  London  County 
Council,  1902.     No.  615.     F.  W.  Andrewes,  M.D.,  F.R.C.P. 


OF  CENTRAL  LONDON  RAILWAY  91 

By  way  of  summary,  it  may  be  said  that  Andrewes  found  that 
micro-organisms  were  present  in  the  air  of  the  Central  London 
Railway  in  a  somewhat  greater  proportion  (as  13  to  10)  than  in  the 
fresh  air  outside.  The  number  was  high  in  proportion  to  the 
concentration  of  human  traffic,  being  highest  in  carriages,  platforms, 
and  lifts.  The  tube  air  does  not  compare  unfavourably  with  that 
known  to  exist  in  ordinary  dwelling-rooms.  No  pathogenic  germs 
were  discovered,  though  the  number  of  organisms  capable  of  growing 
at  body  temperature  was  greater  in  the  tube  air  than  in  the  outside 
air,  and  the  number  of  organisms  in  the  tube  air  was  found  generally 
proportional  to  the  degree  of  chemical  contamination,  but  this  rule 
was  subject  to  striking  exceptions.  It  is  evident  that  bacterial 
contamination  of  air,  though,  as  a  rule,  parallel  to  chemical  con- 
tamination, may  yet  vary  quite  independently  as  the  result  of  special 
conditions,  such  as  air  currents,  which  indicates  that  chemical 
examination  alone  cannot  always  be  taken  as  a  trustworthy  guide  to 
the  contamination  of  air. 

The  species  of  bacteria  which  Andrewes  found  in  the  railway 
air  were  in  the  main  identical  with  those  occurring  in  fresh  air, 
and  included  Staplylococcus  cereus  flavus  et  albus,  Micrococcus  candicans, 
M.  flavus,  M.  citreus,  M.  ladis,  M.  allicans  tardissimus,  Sarcina  lutea, 
S.  flava,  S.  alba,  Bacillus  luteus,  B.  lactis  innocuus,  Streptothrix 
Forsteri,  S.  chromogencs,  S.  albido-flava,  Torula  alba,  and  Saccharomyccs 
cerevisice. 


Interpretation  of  Reports  on  Bacterial  Content  of  Air 

In  the  present  position  of  our  knowledge  of  the  bacteriology  of 
air,  reports  are  only  of  comparative  value.  Mere  numbers  of  sapro- 
phytic  bacteria  in  air  are  not  of  great  service.  Up  to  the  present  it 
has  not  been  possible  to  isolate  pathogenic  organisms,  though  such 
must  inevitably  occur  in  air  under  certain  circumstances,  though  even 
then  probably  only  in  very  small  numbers.  To  detect  pathogenic 
organisms,  it  will  probably  be  necessary  to  examine  large  volumes  of 
air,  and  by  methods  which  will  eliminate  the  common  saprophytes. 
The  truth  is  that  the  foundations  of  our  knowledge  concerning  the 
bacterial  flora  of  the  air  are  only  beginning  to  be  laid,  and  until  we 
can  detect,  by  bacteriological  examination,  organisms  of  disease,  the 
bacteriology  of  air  can  only  be  a  subject  of  relative  importance. 


CHAPTEK  IV 

BACTERIA  AND   FERMENTATION 

Early  Work — Kinds  of  Fermentation:  (1)  Alcoholic  Fermentation,  Ascospores, 
Pure  Cultures,  Films ;  (2)  Acetous  Fermentation ;  (3)  Lactic  Acid  Fermenta- 
tion ;  (4)  Butyric  Fermentation ;  (5)  Ammoniacal  Fermentation — Diseases  of 
Wine  and  Beer:  Turbidity,  Ropiness,  Bitterness,  etc. — Industrial  Applica- 
tions of  Bacterial  Ferments. 

IT  was  Pasteur  who,  in  1857,  first  propounded  the  true  cause  and 
process  of  fermentation.  The  breaking  down  of  sugar  into  alcohol 
and  carbonic  acid  gas  had  been  known,  of  course,  for  a  long  period. 
Since  the  time  of  Spallanzani  (1776)  the  putrefactive  changes  in 
liquids  and  organic  matter  had  been  prevented  by  boiling  and  subse- 
quently sealing  the  flask  or  vessel  containing  the  fluid.  Moreover, 
this  successful  preventive  practice  had  been  in  some  measure 
correctly  interpreted  as  due  to  the  exclusion  of  the  atmosphere,  but 
wrongly  credited  to  the  exclusion  of  the  oxygen  of  the  air.  It  was 
not  until  the  beginning  of  the  present  century  that  authorities  modi- 
fied their  view  and  declared  in  favour  of  yeast  cells  as  the  agents  in 
the  production  of  fermentation.  That  this  process  was  due  to 
oxygen  per  se  was  disproved  by  Schwann,  who  showed  that  so  long 
as  the  oxygen  admitted  to  the  flask  of  fermentable  fluid  was  sterilised 
no  fermentation  occurred.  It  was  thus  obvious  that  it  was  not  the 
atmosphere  or  the  oxygen  of  the  atmosphere,  but  some  fermenting 
agent  borne  into  the  flask  by  the  admission  of  unsterilised  air.  It 
was  but  a  step  further  to  establish  this  hypothesis  by  adding 
unsterilised  air  plus  some  antiseptic  substance  which  would  destroy 
the  fermenting  agent.  Arsenic  was  found  by  Schwann  to  have  this 
germicidal  property.  Hence  Schwann  supported  Latour's  theory 


WORK  OF  PASTEUR  93 

that  fermentation  was  due  to  something  borne  in  by  the  air,  and  that 
this  something  was  yeast. 

Passing  over  a  number  of  counter-experiments  of  Helmholtz  and 
others,  we  come  to  the  work  of  Liebig.  He  viewed  the  transforma- 
tion of  sugar  into  alcohol  and  carbonic  acid  gas  simply  and  solely  as 
a  non-vital  chemical  process,  depending  upon  the  dead  yeast  com- 
municating its  own  decomposition  to  surrounding  elements  in  contact 
with  it.  Liebig  insisted  that  all  albuminoid  bodies  were  unstable, 
and  if  left  to  themselves  would  fall  to  pieces — i.e.  ferment — without 
the  aid  of  living  organisms,  or  any  initiative  force  greater  than 
dead  yeast  cells.  It  was  at  this  juncture  that  Pasteur  intervened  to 
dispel  the  obscurities  and  contradictory  theories  which  had  been 
propounded. 

As  in  all  the  conclusions  arrived  at  by  Pasteur,  so  in  those  relat- 
ing to  fermentation,  there  were  a  number  of  different  experiments 
which  were  performed  by  him  to  elucidate  the  same  point.  We  will 
choose  one  of  many  in  relation  to  fermentation.  If  a  sugary  solution 
of  carbonate  of  lime  is  left  to  itself,  it  begins  after  a  time  to  effervesce, 
carbonic  acid  is  evolved,  and  lactic  acid  is  formed ;  and  this  latter 
decomposes  the  carbonate  of  lime  to  form  lactate  of  lime.  This  lactic 
acid  is  formed,  so  to  speak,  at  the  expense  of  the  sugar,  which  little 
by  little  disappears.  Pasteur  demonstrated  the  cause  of  this  trans- 
formation of  sugar  into  lactic  acid  to  be  a  thin  layer  of  organic 
matter  consisting  of  extremely  small  moving  organisms.  If  these  be 
withheld  or  destroyed  in  the  fermenting  fluid,  fermentation  will 
cease.  If  a  trace  of  this  grey  material  be  introduced  into  sterile  milk 
or  sterile  solution  of  sugar,  the  same  process  is  set  up,  and  lactic 
acid  fermentation  occurs. 

Pasteur  examined  the  elements  of  this  organic  layer  by  aid  of  the 
microscope,  and  found  it  to  consist  of  small  short  rods  of  protoplasm 
quite  distinct  from  the  yeast  cells  which  previous  investigators  had 
detected  in  alcoholic  fermentation.  One  series  of  experiments  was 
accomplished  with  yeast  cells  and  these  bacteria,  a  second  series  with 
living  yeast  cells  only,  a  third  series  with  bacteria  only,  and  the  con- 
clusions at  which  Pasteur  arrived  as  the  result  of  these  labours  he 
expressed  in  the  following  words : — 

"  As  for  the  interpretation  of  the  group  of  new  facts  which  I  have 
met  with  in  the  course  of  these  researches,  I  am  confident  that  who- 
ever shall  judge  them  with  impartiality  will  recognise  that  the 
alcoholic  fermentation  is  an  act  correlated  to  the  life  and  to  the 
organisation  of  these  corpuscles,  and  not  to  their  death  or  their  putre- 
faction, any  more  than  it  will  appear  as  a  case  of  contact  action  in 
which  the  transformation  of  the  sugar  is  accomplished  in  the 
presence  of  the  ferment  without  the  latter  giving  or  taking  anything 
from  it." 


94  BACTERIA  AND  FERMENTATION 

Pasteur  occupied  six  years  (1857-1863)  in  the  further  elucidation 
of  his  discovery  of  the  potency  of  these  hitherto  unrecognised  agents, 
and  the  establishment  of  the  fact  that  "organic  liquids  do  not  alter 
until  a  living  germ  is  introduced  into  them,  and  living  germs  exist 
everywhere."  It  must  not  be  supposed  that  to  Pasteur  is  due  the 
whole  credit  of  the  knowledge  acquired  respecting  the  cause  of 
fermentation.  He  did  not  first  discover  these  living  organisms ;  he 
did  not  first  study  them  and  describe  them ;  he  was  not  even  the 
first  to  suggest  that  they  were  the  cause  of  the  processes  of  fermenta- 
tion or  disease.  But  nevertheless  it  was  Pasteur  who  "  first  placed 
the  subject  upon  a  firm  foundation  by  proving  with  rigid  experiment 
some  of  the  suggestions  made  by  others." 

Kinds  of  Fermentation 

Although  fermentation  is  nearly  always  due  to  a  living  agent,  as 
proved  by  Pasteur,  the  process  is  conveniently  divided  into  two 
kinds.*  (1)  When  the  action  is  direct,  and  the  chemical  changes 
involved  in  the  process  occur  only  in  the  presence  of  the  cell,  the 
latter  is  spoken  of  as  an  organised  ferment ;  (2)  when  the  action  is 
indirect,  and  the  changes  are  the  result  of  the  presence  of  a  soluble 
material  secreted  by  the  cell,  acting  apart  from  the  cell,  this  soluble 
substance  is  termed  an  unorganised  soluble  ferment,  or  enzyme.  The 
organised  ferments  are  bacteria  or  vegetable  cells  allied  to  the 
bacteria ;  the  unorganised  ferments,  or  enzymes,  are  ferments  found 
in  the  secretions  of  specialised  cells  of  the  higher  plants  and  animals. 
It  will  be  sufficient  to  illustrate  the  enzymes  by  a  few  of  the  more 
familiar  examples,  such  as  the  digestive  agents  in  human  assimilation. 
This  function  is  performed,  in  some  cases,  by  the  enzyme  combining 
with  the  substance  on  which  it  is  acting  and  then  by  decomposition 
yielding  the  new  "  digested  "  substance  and  regenerating  the  enzyme ; 
in  other  cases,  the  enzyme,  by  its  molecular  movement,  sets  up 
molecular  movement  in  the  substance  it  is  digesting,  and  thus  changes 
its  condition.  These  digestive  enzymes  are  as  follow :  in  the  saliva, 
ptyalin,  which  changes  starch  into  sugar ;  in  the  gastric  juice  of  the 
stomach,  pepsin,  which  digests  the  proteids  of  the  food  and  changes 
them  into  more  soluble  forms ;  the  pancreatic  ferments,  amylopsin, 
trypsin,  and  steapsin,  capable  of  attacking  all  classes  of  food  stuffs ; 
and  the  intestinal  ferments,  which  have  not  yet  been  separated  in 
pure  condition.  In  addition  to  these,  there  are  ferments  in 
bitter  almonds,  mustard,  etc.  Concerning  these  unorganised  ferments 
we  have  little  further  to  say.  Perhaps  the  commonest  of  them  all 
is  diastase,  which  occurs  in  malt,  and  to  which  some  reference  will  be 
made  later.  Its  function  is  to  convert  the  starch,  which  occurs  in 

*  E.  A,  Schafer,  F.R.S.,  Text-book  of  Physiology,  vol.  i.,  p.  312, 


CONDITIONS  OF  FERMENTATION  95 

barley,  into  sugar.  These  unorganised  ferments  act  most  rapidly  at  a 
high  temperature.* 

We  may  preface  our  consideration  of  the  organised  ferments  by 
an  axiom  by  which  Professor  Frankland  sums  up  the  vitalistic  theory 
of  fermentation,  which  was  supported  by  the  researches  of  Pasteur : 
"  No  fermentation  without  organisms,  in  every  fermentation  a  particular 
organism"  From  these  words  it  is  to  be  inferred  that  there  is  no  one 
particular  organism  or  vegetable  cell  to  be  designated  the  micro- 
organism of  fermentation,  but  that  there  are  a  number  of  fermenta- 
tions each  started  by  some  specific  form  of  agent.  It  is  true  that  the 
chemical  changes,  induced  by  organised  ferments,  depend  on  the  life 
processes  of  micro-organisms  which  feed  upon  the  sugar  or  other 
substance  in  solution,  and  excrete  the  product  of  the  fermentation. 
Fermentation  always  consists  of  a  process  of  breaking  down  of 
complex  bodies,  like  sugar,  into  simpler  ones,  like  alcohol  and 
carbonic  acid.  Of  such  fermentations  we  may  mention  at  least  five : 
the  alcoholic,  by  which  alcohol  is  produced ;  the  acetous,  by  which 
wine  absorbs  oxygen  from  the  air  and  becomes  vinegar ;  the  lactic, 
which  sours  milk ;  the  butyric,  which  out  of  various  sugars  and 
organic  acids  produces  butyric  acid;  and  the  ammoniacal,  which 
is  the  putrefactive  breaking  down  of  compounds  of  nitrogen  into 
ammonia.  We  shall  have  occasion  to  refer  at  some  length  to  this 
process  when  considering  denitrifying  organisms  in  the  soil. 

There  are  four  chief  conditions  common  to  all  these  five  kinds  of 
organised  fermentation.  They  are  as  follow : — 

1.  The  presence  of  the  special  living  agent  or  organism  of  the 
particular    fermentation    under    consideration.      This,    as    Pasteur 
pointed  out,  differs  in  each  case. 

2.  A  sufficiency  of  pabulum  (nutriment)  and  moisture  to  favour 
the  growth  of  the  micro-organism. 

3.  A  temperature  at  or  about  blood-heat  (35-38°  C.,  98'5°  F.). 

4.  The  absence  from  the  solution  or  substance  of  any  obnoxious 
or  inimical  substances  which  would  destroy  or  retard  the  action  of 
the  living  organism  and  agent.     Many  of  the  products  of  fermenta- 
tion are  themselves   antiseptics,  as  in  the  case  of  alcohol;    hence 
alcoholic  fermentation  always  arrests  itself  at  a  certain  point. 

The  causal  micro-organisms  of  particular  fermentations  are  of 
various  kinds,  belonging,  according  to  botanical  classification,  to 

*  The  unorganised  ferments  are  frequently  otherwise  classified  than  as  above, 
according  to  function.  The  chief  are  these  : — amylotytic,  those  which  change  starch 
and  glycogen  (amyloses)  into  sugars,  e.g.,  ptyalin,  diastase,  amylopsin  (organisms 
of  the  subtilis  group  and  the  micrococcus  of  mastitis  are  said  to  produce  amylolytic 
ferments) ;  proteotytic,  those  which  change  proteids  into  proteoses  and  peptones, 
e.g. ,  trypsin,  pepsin ;  inversive,  those  which  change  maltose,  sucrose,  and  lactose 
into  glucose,  e.g. ,  invertin  (various  species  of  bacteria  produce  inversive  ferments) ; 
coayulative,  1:hose  which  change  soluble  proteids  into  insoluble,  e.g.,  rennet ;  steato- 
lytlc,  those  which  split  up  fats  into  fatty  acids  and  glycerine,  e.g.,  steapsin. 


9G  BACTERIA  AND  FERMENTATION 

various  different  subdivisions  of  the  non-flowering  portion  of  the 
vegetable  kingdom.  A  large  part  of  fermentation  is  based  upon  the 
growth  of  a  class  of  microscopic  plants  termed  yeasts.  These  differ 
from  the  bacteria  in  but  few  particulars,  mainly  in  their  method  of 
reproduction  by  budding  (instead  of  dividing  or  sporulating,  like  the 
bacteria).  Their  chemical  action  is  closely  allied  to  that  of  the 
bacteria.  Secondly,  there  are  special  fermentations  and  modifications 
of  yeast  fermentation  due  to  bacteria.  Thirdly,  a  group  of  somewhat 
more  highly  specialised  vegetable  cells,  known  as  moulds,  make  a 
perceptible  contribution  in  this  direction.  According  to  Hansen, 
these  latter,  so  far  as  they  are  really  alcoholic  ferments,  induce 
fermentation,  that  is,  inversion  of  sugar,  not  only  in  solutions  of 
dextrose,  but  also  in  maltose.  Mucor  raeemosus  is  the  only  member 
that  is  capable  of  inverting  a  cane-sugar  solution ;  Mucor  erectus  is 
the  most  active  fermenter,  yielding  8  per  cent,  by  volume  of  alcohol 
in  ordinary  beer  wort.  Both  of  these  will  be  referred  to  as  they 
occur  in  considering  the  five  important  fermentations  already 
mentioned. 

The  general  microscopic  appearance  of  yeast  cells  may  be  shortly 
stated  as  follows :  They  are  round  or  oval  cells,  and  by  budding 
become  "  daughter "  yeast  cells.  Each  consists  of  a  cellulose 
membrane  and  clear  homogeneous  contents.  As  they  perform  their 
function  of  fermentation,  vacuoles,  fat-globules,  and  granules  make 
their  appearance  in  the  enclosed  plasma.  Just  as  in  many  vegetable 
cells  a  nucleus  was  detected  by  Schmitz  by  means  of  special  methods 
of  staining,  so  Hansen  has  found  the  nucleus  in  old-yeast  cells  from 
"  films  "  without  any  special  staining. 

1.  Alcoholic  Fermentation 

Cause,  yeast ;  medium,  sugar  solutions  ;  result,  alcohol  and  carbonic  acid. 

It  was  Caignard-Latour  who  first  demonstrated  that  yeast  cells,  by 
their  growth  and  multiplication,  set  up  a  chemical  change  in  sugar 
solutions  which  resulted  in  the  transference  of  the  oxygen  in  the 
sugar  compound  from  the  hydrogen  to  the  carbon  atoms,  that 
is  to  say,  in  the  evolution  of  carbonic  acid  gas  and  the  production, 
as  a  result,  of  alcohol.  Expressed  in  chemical  formula,  the  change 
is  as  follows : — 

C6H12O6  (plus  the  fermenting  agent)    =    2  C2H0O  +  2  CO2. 

A  natural  sugar,  like  grape-sugar,  present  in  the  fruit  of  the  vine, 
is  thus  fermented.  The  alcohol  remains  in  the  liquid ;  the  carbonic 
acid  escapes  as  bubbles  of  gas  into  the  surrounding  air.  If  we  go 
a  step  further  back,  to  cane-sugar  (which  possesses  the  same  elements 
as  grape-sugar,  but  in  different  proportions),  dissolve  it  in  water, 


ALCOHOLIC  FERMENTATION  97 

and  mix  it  with  yeast,  we  get  exactly  the  same  result,  except  that 
the  first  stage  of  the  fermentation  would  be  the  changing  of  the  cane- 
sugar  into  grape-sugar,  which  is  accomplished  by  a  soluble  ferment 
secreted  by  the  yeast  cells  themselves.  If  now  we  go  yet  one  step 
further  back,  to  starch,  the  same  sort  of  action  occurs.  When  starch 
is  boiled  with  a  dilute  acid  it  is  changed  into  a  gum-like  substance, 
dextrin,  and  subsequently  into  maltose,  which  latter,  when  mixed 
with  these  living  yeast  cells,  is  fermented,  and  results  in  the  evolution 
of  carbonic  acid  gas  and  the  production  of  alcohol.  In  the  manu- 
facture of  fermented  drinks  from  cereal  grains  containing  starch  there 
is,  therefore,  a  double  chemical  process :  first,  the  change  of  starch 
into  sugar  by  means  of  conversion,  a  chemical  change  obtained  by 
the  action  of  sulphuric  or  some  other  acid,  or  by  the  influence  of 
diastase;  and  secondly,  the  change  of  the  sugar  into  alcohol  and 
carbonic  acid  gas  by  the  process  of  fermentation,  an  organic  change 
brought  about  by  the  living  yeast  cells. 

In  all  these  three  forms  of  alcoholic  fermentation  the  principal 
features  are  the  same,  viz.,  the  sugar  disappears ;  the  carbonic  acid 
gas  escapes  into  the  air ;  the  alcohol  remains  behind.  Though  it  is 
true  that  the  sugar  disappears,  it  would  be  truer  still  to  say  that  it 
reappears  as  alcohol.  Sugar  and  alcohol  are  built  up  of  precisely  the 
same  elements:  carbon,  hydrogen,  and  oxygen.  They  differ  from 
each  other  in  the  proportion  of  these  elements.  It  is  obvious,  there- 
fore, that  fermentation  is  really  only  a  change  of  position,  a  breaking 
down  of  one  compound  into  two  simpler  compounds.  And  this 
redistribution  of  the  molecules  of  the  compound  results  in  the 
production  of  some  heat.  Hence,  we  must  add  heat  to  the  results 
of  the  work  of  the  yeasts.* 

It  will  be  necessary  subsequently  to  consider  a  remarkable  faculty 
which  bacteria  possess  of  producing  products  inimical  to  their  own 
growth.  In  some  degree  this  is  true  of  the  yeasts,  for  when  they 
have  set  up  fermentation  in  a  saccharine  fluid  there  comes  a  time 
when  the  presence  of  the  resulting  alcohol  is  injurious  to  further 
action  on  their  part.  It  has  become  indeed  a  poison,  and,  as  we 
have  already  mentioned,  a  necessary  condition  for  the  action  of  a 
ferment  is  the  absence  of  poisonous  substances.  This  limit  of 
fermentation  is  reached  when  the  fermenting  fluid  contains  13  or 
14  per  cent,  of  alcohol. 

The  Biology  of  Yeast. — Having  briefly  discussed  the  "medium" 
and  the  results,  we  may  now  turn  to  the  other  side  of  the  matter, 
and  enumerate  some  of  the  chief  forms  of  the  yeast  plant.  Jorgensen 

"  When  alcohol  is  pure  and  contains  no  water  it  is  termed  absolute  alcohol.  If, 
however,  it  is  mixed  with  16  per  cent,  of  water,  it  is  called  rectified  spirit,  and  when 
mixed  with  more  than  half  its  volume  of  water  (56*8  pej?  cent.)  it  is  known  as  proof 
spirit.  */ 

G 


98 


BACTERIA  AND  FERMENTATION 


gives  more  than  a  score  of  different  members  of  this  family  of 
Saccharomycetes*  But  before  mentioning  some  of  the  chief  of 
these,  it  will  be  desirable  to  consider  a  number  of  properties  common 
to  the  genus.  The  yeast  cell  is  a  round  or  oval  body  of  the  nature 
of  a  fungus,  composed  of  granular  protoplasm  surrounded  by  a 
definite  envelope,  or  capsule.  It  reproduces  itself,  as  a  general 
rule,  by  budding,  or  gemmation.  At  one  end  of  the  cell  a  slight 
swelling  or  protuberance  appears,  which  slowly  enlarges.  Ulti- 
mately there  is  a  constriction,  and  the  bud  becomes  partly  and 
at  last  completely  separated  from  the  parent  cell.  In  many  cases 
the  capsules  of  the  daughter  cell  and  the  parent  cell  adhere,  thus 
forming  a  chain  of  budding  cells.  The  character  of  the  cell  and  its 
method  of  reproduction  do  not  depend  merely  upon  the  particular 
species  alone,  but  are  also  dependent  upon  external  circumstances. 
There  are  differences  in  the  behaviour  of  species  towards  different 
media  at  various  temperatures,  towards  the  carbohydrates  (especially 


FIG.  13. — Diagram  of  Ascospore  Formation. 


FIG.  14. — Gypsum  Block. 


maltose),  and  in  the  chemical  changes  which  they  bring  about  in 
nutrient  liquids.  In  connection  with  these  variations  Professor 
Hansen  has  pointed  out  that,  whilst  some  species  can  be  made  use 
of  in  fermentation  industries,  others  cannot,  and  some  even  produce 
"  diseases  "  in  beer.f 

One  of  the  most  remarkable  evidences  of  the  adaptability  of  the 
yeasts  to  their  surroundings^  and  a  specific  characteristic,  occurs  in 
what  is  termed  ascospore  formation.  If  a  yeast  cell  finds  itself 
lacking  nourishment  or  in  an  unfavourable  medium,  it  reproduces 
itself  not  by  budding,  but  by  forming  spores  out  of  its  own  intrinsic 
substance,  and  within  its  own  capsule.  To  obtain  this  kind  of 
spore  formation  Hansen  used  small  gypsum  blocks  as  the  medium  on 
which  to  grow  his  yeast  cells.  Well-baked  plaster-of-Paris  is  mixed 
with  distilled  water,  and  made  into  a  liquid  paste.  The  moulds  are 
made  by  pouring  this  paste  into  cardboard  dishes,  where  it  hardens 

*  Micro-organisms  and  Fermentation. 

f  E.  C.  Hansen,  Studies  in  Fermentation  (Copenhagen),  p.  98. 


PLATE  8. 


SacchciTomyces  cerevisice. 
Film  preparation,     x  1000. 


ASCOSPORE  FORMATION  IN  YEAST.  The  capsule 
of  the  parent  cell  around  the  spores  is 
invisible,  x  1000. 


V 


I 


<P 


o 


CO 


0° 

O  MS  0  '  t) 


co 


PATHOGKNIC  YEAST.    (Foulerton).     x  1000. 


[To  face  page  98. 


ALCOHOLIC  FERMENTATION  99 

again.  The  mould  is  then  sterilised  by  heat,  a  few  cells  of  yeast  are 
placed  on  its  upper  surface,  and  the  whole  is  floated  in  a  small 
vessel  of  water  and  covered  with  a  bell-jar.  Under  these  conditions 
of  limited  pabulum  the  cell  undergoes  the  following  changes:  it 
increases  in  size,  loses  much  of  its  granularity,  and  becomes  homo- 
geneous, and  about  thirty  hours  after  being  sown  on  ths  gypsum 
there  appear  several  refractile  cells  inside  the  parent  cell.  These 
are  the  ascospores.  In  addition  to  the  gypsum,  it  is  necessary  to 
have  a  plentiful  supply  of  oxygen,  some  moisture  (gained  from  the 
vessel  of  water  in  which  the  gypsum  stands),  a  certain  temperature, 
and  a  young  condition  of  the  protoplasm  of  the  parent  yeast  cells. 
Hansen  found  that  the  lowest  temperature  at  which  these  ascospores 
were  produced  was  *5  —  3°  C.,  and  at  the  other  extreme  up  to  37°  C. 
The  rapidity  of  formation  also  varies  with  the  temperature,  the 
favourable  degree  of  warmth  being  about  22  —  25°  C.  (Plate  8). 

Hansen  pointed  out  that  it  was  possible  by  means  of  sporulation 
to  differentiate  species  of  yeasts.  For  it  happens  that  different 
species  show  slight  differences  in  spore  formation,  e.g. : — 

(a)  The  spores  of  Saccharomyces  cerevisice  expand  during  the  first 
stage  of  germination,  and  produce  partition  walls,  making  a  com- 
pound cell  with  several  chambers.  Budding  can  occur  at  any  point 
on  the  surface  of  the  swollen  spores.  To  this  group  belong  S. 
pastorianus  and  8.  ellipsoideus. 

(5)  The  spores  of  Saccharomyces  Ludwigii  fuse  in  the  first  stage,  and 
afterwards  grow  out  into  a  promycelium,  which  produces  yeast  cells. 

(c)  The  spores  of  Saccharomyces  anomalus  are  different  in  shape 
from  the  others  in  that  they  possess  a  projecting  rim  round 
the  base. 

Another  point  in  the  cultivation  of  yeasts  has  been  elucidated  by 
a  number  of  workers,  among  whom  is  Hansen,  namely,  the  methods 
of  obtaining  pure  cultures.  Only  by  starting  with  one  individual 
cell  can  it  be  hoped  to  secure  a  pure  culture  of  yeasts.  For  the 
study  of  the  morphology  of  yeasts  under  the  microscope  the  problem 
was  not  a  difficult  one.  It  was  comparatively  easy  to  keep  out 
foreign  germs  from  a  cover-glass  preparation  sufficiently  to  perceive 
germination  of  spores  and  the  growth  of  yeasts.  But  when  pure 
cultures  are  required  for  various  physiological  purposes  then  a 
different  standard  and  method  is  necessary. 

Hansen  employed  dilution  with  water  in  the  following  manner : — 
Yeast  is  diluted  with  a  certain  amount  of  sterilised  water.  A  drop 
is  carefully  examined  under  the  microscope,  a  single  cell  of  yeast  is 
taken,  and  a  cultivation  made  upon  wort.  When  it  has  grown 
abundantly  a  quantity  of  sterilised  water  is  added.  From  this, 
again,  a  single  drop  is  taken  and  added  to  say  20  c.c.  of  sterilised 
water  in  a  fresh  flask.  This  flask  will  contain,  let  us  suppose,  ten 


100  BACTERIA  AND  FERMENTATION 

cells.  It  is  now  vigorously  shaken,  and  the  contents  are  divided 
into  twenty  portions  of  1  c.c.  each,  and  added  to  twenty  tubes  of 
sterilised  water.  It  is  highly  probable  that  half  of  these  tubes 
have  received  one  cell  each.  In  the  course  of  a  few  days  it  can  be 
seen  how  far  the  culture  is  pure.  If  only  one  colony  is  present, 
the  culture  is  a  pure  one,  and  as  this  grows  we  obtain  an  absolutely 
pure  culture  in  necessary  quantity.  Even  when  the  gelatine  plate 
method  is  used,  it  is  desirable  to  start  with  a  single  cell  (Hansen). 
The  advantage  of  Hansen's  yeast  method  over  Koch's  bacterial  plate 
method  is  that  it  has  a  certain  definite  starting-point.  This  is 
obviously  impossible  when  dealing  with  such  microscopic  particles 
as  the  bacteria  proper. 

A  third  point  in  the  differentiation  of  yeast  species  is  the  question 
of  films.  Hansen  set  to  work,  after  having  obtained  pure  cultures 
and  ascospores,  to  examine  films  appearing  on  the  surface  of  liquids 
undergoing  fermentation.  The  object  of  this  was  to  ascertain 
whether  all  yeasts  produced  the  same  mycelial  growth  on  the  surface 
of  the  fermenting  fluid.  To  produce  these  films  the  process  is  as 
follows :  Drop  on  to  the  surface  of  sterilised  wort  in  a  flask  a  very 
small  quantity  of  a  pure  culture  of  yeast ;  secure  the  flask  from 
movement,  and  protect  it,  not  from  air,  which  is  necessary,  but  from 
falling  particles  in  the  air.  In  a  short  time  small  colonies  appear, 
which  coalesce  and  form  patches,  and  finally  a  film  or  membrane 
which  covers  the  liquid  and  attaches  itself  to  the  sides  of  the  flask. 
By  the  differences  in  the  films  and  the  temperatures  at  which  they 
form  it  is  possible  to  obtain  something  of  a  basis  for  classification. 
The  further  advances  in  yeast  culture  and  in  our  knowledge  of  the 
agencies  of  fermentation  have,  however,  tended  to  show  that  no 
strict  dividing  lines  can  be  drawn.  Hansen's  researches  have,  not- 
withstanding, been  of  the  greatest  moment  to  the  whole  industry  of 
fermentation.  What  has  been  found  true  in  bacteriology  has  also 
been  demonstrated  in  fermentation,  namely,  that,  though  many 
yeasts  differ  but  little  in  structure  and  behaviour,  they  may  produce 
very  different  products  and  possess  very  different  properties. 
Industrial  cultivation  of  these  finer  differences  in  fermentative  action 
has  to  a  large  extent  revolutionised  the  brewing  industry.  The 
formation  of  films  is  not  a  peculiarity  of  certain  species,  but  must 
be  regarded  as  a  phenomenon  occurring  somewhat  commonly  amongst 
yeasts.  The  requisites  are  a  suitable  medium,  a  yeast  cell,  a  free, 
still  surface,  direct  access  of  air,  and  a  favourable  temperature.  The 
wort  loses  colour,  and  becomes  pale  yellow.  Microscopic  differences 
soon  appear  between  the  sedimentary  yeast  and  the  film  yeast  of  the 
same  species,  the  latter  growing  out  into  long  mycelial  forms,  the 
character  of  which  depends  in  part  upon  the  temperature.  This 
often  varies  from  3°  to  38°  C. 


ALCOHOLIC  FERMENTATION  101 

A  fourth  point  helpful  in  diagnosis  is  the  temperature  which 
proves  to  be  the  thermal  death-point.  SaccJiaromyces  cerevisice  is 
killed  by  an  exposure  to  54°  C.  for  five  minutes,  and  62°  C.  kills  the 
spores.  As  a  rule,  yeasts  can  resist  a  considerably  higher  tempera- 
ture when  in  a  dry  state  than  in  the  presence  of  moisture. 

It  should  be  noted  that  yeasts  may  be  cultivated  on  solid 
media.  Hansen  employed  wort-gelatine  (5  per  cent,  gelatine),  and 
found  that  at  25°  C.  in  a  fortnight  the  growths  which  develop  show 
such  microscopic  differences  as  to  aid  materially  in  diagnosis. 
Saccliaromyces  ellipsoideus  I.  exhibits  a  characteristic  network  which 
readily  distinguishes  it. 

There  is  one  other  feature  to  which  reference  must  be  made. 
The  process  of  fermentation  may  be  set  up  by  a  "high"  or  a 
"  low "  yeast.  These  terms  apply  to  the  temperature  at  which  the 
process  commences.  "  High  "  yeasts  rise  to  the  surface  as  the  action 
proceeds,  accomplish  their  work  rapidly,  and  at  a  comparatively  high 
temperature,  say  about  16°  C. ;  "low"  yeasts,  on  the  contrary,  sink 
in  the  fermenting  fluid,  act  slowly,  and  only  at  the  low  temperature 
of  4°  or  5°  C.  This  is  maintainable  by  floating  ice  in  the  fluid. 
Formerly  all  beer  was  made  by  the  "  high "  mode,  but  on  the  con- 
tinent of  Europe  "  low  "  yeast  is  mostly  used,  whilst  the  '•  high  "  is  in 
vogue  in  England.  This  latter  method  is  more  conducive  to  the 
development  of  extraneous  organisms,  and  therefore  risky  in  all  but 
well-ordered  brewing  establishments. 

Before  proceeding  to  mention  shortly  some  of  the  commoner 
forms  of  yeast  we  must  again  emphasise  Hansen's  method  of  analysis 
in  separating  a  species.  The  shape,  size,  and  appearance  of  cells  are 
not  sufficient  for  differentiation,  because  it  is  found  that  the  same 
species,  when  exposed  to  different  external  conditions,  can  occur  in 
very  different  forms.  Hence  Hansen  established  the  analytical 
method  of  observing  (1)  the  microscopic  appearance,  (2)  the  forma- 
tion of  ascospores,  and  (3)  the  production  of  films.  In  addition,  the 
temperature  limits,  cultivation  on  solid  media,  and  behaviour  towards 
carbohydrates,  are  characters  which  aid  in  the  separation  of  yeasts. 
In  well-grown  cultures  on  wort-gelatine,  a  broad  division  can  be 
made  of  yeasts  according  as  they  produce  (a)  a  dry,  hard,  cohesive 
growth ;  (b)  a  soft,  moist  growth  with  liquefaction  of  gelatine ;  and 
(c)  those  producing  pigment.  By  basing  differentiation  of  species 
upon  these  features,  the  following  can  be  distinguished : — 

Saccliaromyces  Cerevisice.— Oval  or  ellipsoidal  cells;  reproduction  by  budding; 
ascospores,  rapidly  at  30°  C.,  slowly  at  12°  C.,  not  formed  at  all  at  lower  tempera- 
tures ;  film  formation,  seven  to  ten  days  at  22°  C.  ;  an  active  alcoholic  ferment, 
producing  in  a  fortnight  in  beer  wort  from  4  to  6  per  cent,  by  volume  of  alcohol 
(Jorgensen).  This  species  is  a  typical  English  high  yeast,  possessing  the  power  of 
"  inverting  "  cane  sugar  previous  to  producing  alcohol  and  carbonic  acid.  It  is  said 
to  have  no  action  on  milk-sugar.  It  is  the  "  true  brewing  yeast "  (Plate  8). 


102 


BACTERIA  AND  FERMENTATION 


Saccharomyces  Ellipsoidus  I. — Round,  oval,  or  sausage-shaped  cells,  single  or  in 
chains;  ascospores  in  twenty-four  hours  at  25°  C.  (not  above  30°  C. ,  not  below 
4°  C.).  Grown  on  the  surface  of  wort  gelatine,  a  network  is  produced  by  which 
they  can  be  recognised  (in  eight  to  twelve  days  at  33°  C.).  At  13-15"  C.  a 
characteristic  branching  mass  is  produced.  It  is  an  alcoholic  ferment  as  active  as 


O     00 


FIG.  15.—  Diagram  of  S.  cerevisite. 


FIG.  16.— Diagram  of  S.  cllipsoideus. 


S.  cerevisice.  S.  ellipsoideus  II. — Round  and  oval,  rarely  elongated,  a  widely  dis- 
tributed yeast,  causing  **  muddiness  "  in  beer  and  a  bitter  taste.  It  is  essentially  a 
"  low  "  yeast,  and  one  of  the  so-called  "  wild  yeasts  "  injurious  to  beer. 

Saccharomyces   Conglomerate  is  a  round  cell,   often   united  in  clusters,   and 
occurring  in  rotting  grapes,  and  at  the  commencement  of  fermentation. 

Saccharomyces  Pastorianus  I. — Oval   or  club-shaped  cells,   occurring  in   after- 
fermentation   of  wine,  etc.,  and  producing  a  bitter  taste,  unpleasant  odour,  and 
turbidity.     The  spores  frequently  occur  in  the  air  of 
breweries. 

S.  Pastor.  II. — Elongated  cells,  possessing  an  in- 
vertose  ferment.  They  do  not,  like  S.  pastor.  I., 
produce  disease  in  beer. 

S.  Pastor.  111. — Oval  or  elongated  cells,  producing 
turbidity  in  beer.  Grown  on  yeast-water  gelatine ; 
the  colonies  show  after  sixteen  days  crenated  hairy 
edges. 

Saccharomyces  Apiculatus.  —  Lemon-shaped  cells. 
They  give  rise  to  a  feeble  alcoholic  fermentation,  and 
produce  two  kinds  of  spores— round  and  oval ;  they 
appear  at  the  onset  of  vinous  fermentation,  but  give 
way  later  on  to  S.  cerevisice. 

Saccharomyces  Mycoderma. — Oval  or  elliptical  cells, 
often  in  branching  'chains.  They  form  the  so-called 
"mould"  on  fermented  liquids,  and  develop  on  the 
surface  without  exciting  fermentation.  When  forced  to  grow  submerged,  they 
produce  a  little  alcohol. 

Saccharomyces  Exiyuus.  —Conical  cells,  appearing  in  the  after-fermentation  of 
beer. 

Saccharomyces  Pyriformis.—Oval  cells,  converting  sugary  solutions  containing 
ginger  into  ginger-beer. 

Saccharomyces  lllicis,  Hansenii,  and  Aquifolii  produce  a  small  percentage  of 
alcohol. 

2.  Acetous  Fermentation 

Cause,   Mycoderma   aceti ;  medium,  wine  and  other  alcoholic  liquids;   result,  the 

formation  of  vinegar. 

If  alcohol  be  diluted  with  water,  and  the  specific  ferment  mixed 
with  it  and  exposed  to  the  air  at  22°  C.,  it  is  rapidly  converted  into 


FIG.  17.— Diagram  of  S. 
pastorianus. 


ACETOUS  FERMENTATION  103 

vinegar.  The  change  is  accompanied  by  the  absorption  of  oxygen, 
one  atom  of  which  combines  with  two  of  hydrogen  to  form  water,  and 
a  substance  remains  termed  aldehyde,  further  oxidation  of  which  pro- 
duces the  acetic  acid.  We  may  express  it  chemically  thus : — 

C2H6O  (  + oxygen  and  the  ferment)    =    C2H4O    +    H2O. 

Alcohol.  Aldehyde.  Water. 

The  aldehyde  becomes  further  oxidised : — 

C2H4O  +  O    =    C2H4O2  (acetic  acid). 

This  method  of  simply  oxidising  alcohol  to  obtain  acetic  acid  may 
be  carried  out  chemically  without  any  ferment.  If  slightly  diluted 
alcohol  be  dropped  upon  platinum  black,  the  oxygen  condensed  in 
that  substance  acts  with  energy  upon  the  spirit,  and  union  readily 
occurring,  acetic  acid  results.  Here  the  whole  business  of  the  plati- 
num sponge  is  to  persuade  the  oxygen  of  the  air  and  the  hydrogen  of 
the  alcohol  to  unite.  In  the  ordinary  manufacture  this  is  accom- 
plished by  the  vegetable  cells  of  Mycoderma  aceti. 

There  are  two  chief  methods  adopted  in  the  commercial  manu- 
facture of  vinegar,  both  of  which  depend  upon  the  presence  of  the 
mycodcrma.  The  method  in  vogue  at  Orleans  when  Pasteur  (about 
1862)  commenced  his  studies  of  the  vinegar  organism,  was  to  fill  vats 
nearly  to  the  brim  with  a  weak  mixture  of  vinegar  and  wine.  Where 
the  process  is  proceeding  the  surface  is  covered  with  a  fragile  pellicle, 
"the  mother  of  vinegar,"  which  is  produced  by,  and  consists  of, 
certain  micro-organisms  whose  function  is  to  convey  the  oxygen  of 
the  air  to  the  liquor  in  the  vats,  thus  oxidising  the  alcohol  into 
vinegar.  This  oxidation  may  be  carried  on  even  beyond  the  stage  of 
acetic  acid  (when  no  more  alcohol  remains  to  be  oxidised),  resulting 
in  carbonic  acid  gas,  which  escapes  into  the  air.  But  as  in  the 
alcoholic,  so  in  the  acetic,  fermentation  there  comes  a  time  when  the 
presence  of  an  excess  of  the  acid  inhibits  the  further  growth  of  the 
organism.  This  point  is  approximately  when  the  acetic  acid  has 
reached  a  percentage  as  high  as  14.  But  if  the  acid  be  removed,  and 
fresh  alcohol  added,  the  process  recommences. 

The  second  method,  sometimes  called  by  the  Germans  the  "  quick 
vinegar  process,"  is  to  pour  the  weakened  alcohol  through  a  tall 
cylinder  filled  with  wood-shavings,  having  first  added  some  warm 
vinegar  to  the  shavings.  After  a  number  of  hours  the  resulting  fluid 
is  charged  with  acetic  acid.  What  has  occurred  ?  Liebig  maintained 
that  a  chemical  and  mechanical  change  had  brought  about  the  change 
from  the  alcohol  put  into  the  cylinder  and  the  vinegar  drawn  off  at 
the  exit  tube.  It  was  reserved  for  Pasteur  to  demonstrate  by  experi- 
ment that  the  addition  of  the  warm  vinegar  to  the  shavings  was  in 
reality  an  addition  of  a  living  micro-organism,  which,  forming  a  film 


104  BACTERIA  AND  FERMENTATION 

upon  the  shavings,  became  "  the  mother  of  vinegar,"  and  oxidised  the 
alcohol  which  passed  over  it,  inducing  it  to  become  aldehyde  and  then 
acetic  acid. 

Mycoderma  aceti  (described  by  Persoon  1822,  Kiitzing  1837,  and 
Pasteur  1864),  is  the  name  rather  of  a  family  than  an  individual. 
Pasteur  believed  it  to  be  a  specific  individual,  but  Hansen  pointed 
out  that  it  was  composed  of  two  distinctly  different  species  (Bacterium 
aceti  and  B.  Pasteur ianum),  and  subsequently  other  investigators 
have  added  members  to  the  acetic  fermentation  group  of  which  M. 
aceti  is  the  type.  This  bacterium  is  made  up  of  small,  slightly 
elongated  cells,  with  a  transverse  diameter  of  2  or  3  /m,  sometimes 
united  in  short  chains  of  curved  rods.  They  frequently  show  a 
central  constriction,  are  motile,  and  produce  in  old  cultures  involution 
forms.  The  way  in  which  the  cells  act  and  are  made  to  perform  their 
function  is  as  follows:  A  small  quantity,  taken  from  a  previous 
pellicle,  is  sown  on  the  surface  of  an  aqueous  liquid,  containing  2  per 
cent,  of  alcohol,  1  per  cent,  of  vinegar,  and  traces  of  alkaline  phos- 
phates. Very  rapidly  indeed  the  little  isolated  colonies  spread,  and 
becoming  confluent,  form  a  membrane  or  pellicle  over  the  whole  area 
of  the  fluid.  When  the  surface  is  covered  the  alcohol  is  converted  to 
acid.  After  this  it  is  necessary  to  add,  each  day,  small  quantities  of 
alcohol.  When  the  oxidation  is  completed  the  vinegar  is  drawn  off, 
and  the  membrane  is  collected  and  washed,  and  is  then  again  ready 
for  use.  It  ought  not  to  remain  long  out  of  fermenting  liquid,  nor 
ought  it  to  be  allowed  to  over-perform  its  function,  for  thus  having 
oxidised  all  the  alcohol  it  will  commence  oxidation  of  the  vinegar. 

In  wort-gelatine  Bacterium  Pasteurianum  develops  as  round 
colonies  with  a  smooth  or  wavy  border,  whilst  B.  aceti  has  a  tendency 
towards  stellate  arrangement.  Spores  have  not  been  observed,  and 
from  a  morphological  point  of  view  the  two  species  behave  alike. 
Neither  produces  any  turbidity  in  the  liquid  containing  them.  In 
order  to  flourish,  B.  aceti  requires  a  temperature  of  about  33°  C.  and 
a  plentiful  supply  of  oxygen.  In  a  cool  store  or  cellar  there  is, 
therefore,  nothing  to  fear  from  B.  aceti.  Frankland  has  isolated  a 
Bacillus  ethaceticus,  which  is  a  fermentative  organism  producing 
ethyl-alcohol  and  acetic  acid.  By  oxidation  the  ethyl-alcohol  may  be 
converted  into  acetic  acid. 


3.— Lactic  Acid  Fermentation 

Cause,  Bacillus  acidi  lactlci;  medium,  milk-sugar,  cane-sugar,  glucose,  dextrose, 
etc.  ;  result,  lactic  acid. 

The  process  set  up  by  the  lactic  ferment  is  simply  a  decomposition, 
an  exact  division  of  one  molecule  of  sugar  into  two  molecules  of 
lactic  acid,  there  being  neither  oxidation  nor  hydration.  The  con- 


LACTIC  ACID  FERMENTATION  105 

ditions  under  which  the  ferment  acts  are  very  similar  to  those  we 
have  already  considered  (see  also  p.  196).  There  is  frequently  car- 
bonic acid  gas  formed ;  there  is  a  cessation  of  fermentation  when  the 
medium  becomes  too  acid ;  there  is  the  same  method  of  starting  the 
process  by  inoculation  of  milk  or  cheese  or  any  such  substance  with 
the  specific  bacillus.  It  is  probable  that  such  inoculated  matter  will 
contain  a  mixture  of  micro-organisms,  but  if  the  lactic  bacillus  is 
present,  it  will  grow  so  vigorously  and  abundantly  that  the  fermen- 
tation will  be  readily  set  up.* 

In  1877  Lister  was  able,  by  means  of  the  "  dilution  method,"  to 
isolate  from  sour  milk,  in  a  form  of  pure  culture,  an  organism  to 
which  he  gave  the  name  B.  lactis,  and  which  he  believed  gained 
access  to  milk  from  the  air  of  dairies  and  similar  places.f  For  some 
time  this  organism  was  held  to  be  causally  related  to  lactic 
fermentation.  But  in  1884,  by  means  of  culture  on  solid  media,  as 
introduced  by  Koch,  Hueppe  was  able  to  isolate  a  bacillus  which  he 
named  the  Bacillus  acidi  lactici.  This  was  probably  identical  with 
Lister's  bacillus,  and  is  now  a  term  used  to  cover  a  whole  family  of 
organisms  having  somewhat  similar  characters,  and  possessing  the 
property  of  setting  up  lactic  fermentation.^  In  1894  Giinther  and 
Thierfelder  published  the  result  of  .their  work  on  lactic  acid 
fermentation,  from  which  they  concluded  that  Lister  and  Hueppe 
had  discovered  one  and  the  same  species,  and  that  it  was  the  causal 
agent  of  lactic  acid  production  in  Europe.  Esten  found  a  similar 
organism  to  be  the  cause  of  lactic  acid  fermentation  in  America,  and 
Conn  holds  that  three  organisms,  or  rather  types  of  species,  are  the 
chief  agents  in  the  production  of  lactic  fermentation,  namely  B.  acidi 
lactici,  Nos.  L  and  ii.,  and  B.  lactis  ccrogenes.  The  first  named  forms 
between  75  to  90  per  cent,  of  the  bacteria  present.  No.  ii.  is  also 
very  abundant.  B.  lactis  cerogenes  is  found  almost  universally, 
although  never  in  large  numbers.  It  is  a  type  of  a  species  which 
produces  intense  acid  on  litmus  gelatine  cultures,  produces  much 
gas  in  milk  or  milk-sugar  broth,  curdles  milk  at  high  temperatures, 
and  produces  a  distinctive  odour  in  the  milk,  which  it  ferments.  § 
According  to  Escherich,  the  formation  of  lactic  acid  by  this 
organism  prevents  fermentation  in  the  stomach  and  intestines. 

It  was  Hueppe  who  made  the  important  discovery  that  many 

*  For  full  discussion  of  the  subject  of  lactic  fermentation  of  milk,  see  Bacteriology 
of  Milk,  1903  (Swithinbank  and  Newman),  pp.  149-159. 

t  Trans,  of  Path.  Soc.,  1878,  p.  437. 

I  Hueppe  isolated  five  forms  of  his  lactic  acid  bacillus,  and  Fliigge  described 
eleven  forms.  Maddox,  Beyer,  Fokker,  Krueger,  Grotenfeld,  and  other  workers 
isolated  lactic  acid  organisms. 

§  Storr's  Agricultural  Expt.  Sta.  Rep.,  1899,  p.  22.  Others  than  those  named 
are  B.  acidi  lactici  of  Giinther,  B.  acidi  lactici  of  Leichmann,  Bacillus  XIX.  of 
Adametz,  Bacillus  a.  of  Freudenreich,  B.  and  M.  acidi  la^volactici  of  Leichmann, 
Grotenfeld's  B.  acidi  lactici  (Nos.  i.  and  ii.),  No.  8  of  Eckles,  and  B.  casei. 


106  BACTERIA  AND  FERMENTATION 

different  species  of  bacteria  are  capable  of  setting  up  lactic  fer- 
mentation, and  what  we  have  now  said  amply  supports  that  view. 
Indeed,  it  has  been  estimated  that  upwards  of  100  different  bacteria 
possess  this  property.* 

Bacillus  acidi  lactici  (Hiippe)  consists  of  rods  about  2  fj,  long  and  '4  /x,  wide, 
occurring  singly  or  in  pairs,  chains  or  threads.  Its  habitat  is  sour  milk. 

It  grows  best  at  blood-heat,  but  much  above  that  it  fails  to  produce  its  fermenta- 
tion. It  ceases  to  grow  under  10°  C.  It  inverts  milk-sugar  and  changes  it  to 
dextrose,  from  which  it  then  produces  lactic  acid.  Sugars  do,  however,  differ 
considerably  in  the  degrees  to  which  they  respond  to  the  influence  of  the  lactic 
ferment,  and  some  which  are  readily  changed  by  the  alcoholic  ferment  are  un- 
touched by  Bacillus  acidi  lactici. 

Staining  reaction — Ordinary  stains  and  slightly  by  Gram's  method. 

Motility—No  flagella ;  non-motile. 

Spore  formation — Absent. 

Biology:  cultural  characters  (including  biochemical  features) — Good  growth  at 
room  temperature  and  blood-heat. 

Bouillon — Diffuse  turbidity  ;  abundant  sediment. 

Gelatine  plates  and  tubes — Colonies  similar  to  B.  coli;  small,  smooth,  round, 
white. 

Agar  plates  and  tubes — Colonies  similar  to  B.  coli;  small,  smooth,  white  growths, 
moist  and  shiny. 

Potato — A  wavy,  smooth-edged  growth,  elevated  ;  greyish-white  or  yellowish- 
white  in  colour,  sometimes  turning  brown. 

Milk — Solid  coagulation,  leaving  clear  fluid ;  occasionally  some  gas-bubbles. 
Lactic  and  acetic  acids  are  produced.  Powers  of  acid  coagulation  of  milk  are 
gradually  lost  after  long  cultivation  upon  gelatine  or  agar. 

Anaerobic  or  aerobic — Grows  well  aerobically,  and  if  sugar  present  in  medium 
anaerobically  also. 

Non- pathogenic. 

The  lactic  fermentation  bacteria  are  short  rods,  do  not  liquefy 
gelatine,  nor  do  they  form  spores.  They  grow  readily  on  gelatine  at 
room  temperature,  forming  as  a  rule  small  circular  colonies,  white  or 
grey  in  colour,  with  sometimes  a  tinge  of  yellow,  and  the  surface  of 
the  colony  is  smooth  and  glistening.  The  lactic  acid  organisms  pro- 
duce appreciable  amounts  of  lactic  acid  only  at  somewhat  elevated 
temperatures.  If  the  amount  of  acid  rises  much  above  2  per  cent., 
the  growth  of  the  lactic  acid  bacteria  is  inhibited.  Many  other  sub- 
stances, as  we  have  seen,  are  produced  in  addition  to  lactic  acid  (e.g. 
acetic  and  ferric  acids,  alcohol,  methane,  C02 .  H .  N".,  etc.).  Lactic  acid 
organisms  (as  non-spore  bearers)  are  readily  killed  by  pasteurisation. 

*  Delbriick,  Zopf,  Krause,  Peters,  Lindner,  Weigmann,  Storch,  and  Marpmann, 
are  amongst  those  who,  in  addition  to  workers  we  have  named,  have  described 
bacteria  possessing  the  power  of  setting  up  lactic  fermentation.  Only  provisional 
classifications  are  possible  at  present,  as,  owing  to  variations  in  biology  and 
terminology,  it  is  probable  that  certain  lactic  organisms  are  described  under  several 
different  terms.  Generally,  it  may  be  said  that  some  grow  well  in  the  presence  of 
oxygen,  and  others  do  not.  The  latter  group,  facultative  anaerobes,  are  perhaps 
the  most  common.  They  sour  milk  best  in  deep  vessels,  and  produce  a  right- 
handed  lactic  acid.  They  are  widely  distributed  in  nature,  and  may  form  90  per 
cent,  of  the  total  bacteria  in  milk.  Some  produce  gas,  others  liquefy  gelatine,  and 
yet  others  produce  spores. 


BUTYRIC  ACID  FERMENTATION  107 

The  economic  function  of  the  lactic  ferments  concerns,  of  course,  the 
manufacture  of  butter  and  cheese. 

Van  Laer  has  described  a  saccharo-bacillus  which  produces  lactic 
acid  amongst  other  products,  and  brings  about  a  characteristic 
disease  in  beer,  named  tourne.  The  liquid  gradually  loses  its  bright- 
ness and  assumes  a  bad  odour  and  disagreeable  taste.  The  bacillus 
is  a  facultative  anaerobe.  A  number  of  workers  have  separated 
organisms  having  a  lactic  acid  effect,  which  diverge  considerably  from 
the  ordinary  type  of  lactic  acid  bacillus. 

4.  Butyric  Acid  Fermentation 

Cause,  Bacillus  butyricus  and  other  forms ;   medium,  milk,  butter,  sugar  and 
starch  solutions,  glycerine;  result,  butyric  acid. 

When  sugars  are  broken  down  by  Bacillus  acidi  lactici  the  lactic 
acid  resulting  may,  under  the  influence  of  the  butyric  ferment 
become  converted  into  butyric  acid,  carbonic  acid,  and  hydrogen. 
Neither  butyric  acid  nor  lactic  acid  is  as  commonly  used  as  alcohol 
or  vinegar.  Both,  like  vinegar,  can  be  manufactured  chemically, 
but  this  is  rarely  practised.  Butyric  acid  is  a  common  ingredient 
in  stale  milk  and  butter,  and  its  production  by  bacteria  was 
historically  one  of  the  first  bacterial  fermentations  understood. 
Moreover,  in  its  investigation  Pasteur  first  brought  to  light  the 
fact  that  certain  organisms  acted  only  in  the  absence  of  oxygen. 
In  studying  a  drop  of  butyric  fermenting  fluid,  it  was  observed 
that  the  organisms  at  the  edge  of  the  drop  were  motionless  and 
apparently  dead,  whilst  in  the  central  portion  of  the  drop  the 
bacilli  were  executing  those  active  movements  which  are  character- 
istic of  their  vitality.  To  Pasteur's  mind  this  at  once  suggested 
what  he  was  able  later  to  demonstrate,  namely,  that  these  bacilli 
were  paralysed  by  contact  with  oxygen.  When  he  passed  a  stream 
of  air  through  a  flask  containing  a  liquid  in  butyric  fermentation, 
he  observed  the  process  slacken  and  eventually  cease.  .So  were 
discovered  the  anaerobic  micro-organisms.  The  aerobic  ferments 
give  rise  to  oxidation  of  certain  products  of  decomposition;  the 
anaerobic  organisms,  on  the  other  hand,  only  commence  to  grow 
when  the  aerobic  have  used  up  all  the  available  oxygen.  Thus  in 
such  fermentations  certain  bodies  (carbohydrates,  fatty  acids,  etc.) 
undergo  decomposition,  and  by  oxidation  become  carbonic  acid  gas, 
and  the  remainder  is  left  as  a  "  reduced "  product  of  the  whole 
process.  Hence  sometimes  this  is  termed  fermentation  by  reduction. 
The  chemical  formula  of  this  butyric  reaction  may  be  expressed 
thus  : — 

Ci;H12O0  (by  simple  decomposition)    =    2  C3H0O3, 

Glucose.  Lactic  acid. 


108  BACTERIA  AND  FERMENTATION 

which  is  followed  by  the  fermentation  of  the  lactic  acid: — 

2C3H6O3        =        C4H8O2        +        2CO2        +        2  H2. 

Lactic  acid.  Butyric  acid.  Carbonic  Free  hydrogen. 

acid  gas. 

Previously  to  1880,  the  only  work  which  had  been  done  in  the 
elucidation  of  the  bacterial  origin  of  butyric  fermentation  had  been 
accomplished  by  Prazmowski  and  Pasteur;  the  former  designating 
the  organism  he  found  Clostridium  lutyricum,  and  the  latter  naming 
his  "infusoires"  Vibrion  lutyrique.  Prazmowski  emphasised  the 
motility  and  resistance  of  the  bacillus,  and  found  that  the  latter  was 
due  to  the  spores  produced  by  the  organism.  These  spores  were 
able  to  withstand  boiling  for  several  minutes.  Fitz  went  so  far  as 
to  say  that  butyric  spores  could  resist  boiling  for  twenty  minutes. 
Prazmowski  was  unable  to  obtain  pure  cultures.  Clostridium  buty- 
ricum  grows  most  readily  at  a  temperature  of  about  40°  C.,  and  is 
very  widely  distributed  in  nature.  It  is  capable  of  dissolving  cellu- 
lose, and  therefore  plays  a  part  in  the  cellulose  fermentation,  which 
is  employed  in  various  maceration  industries.  It  is  generally  held 
that  in  such  fermentations  there  is  symbiotic  action  between  the 
butyric  bacillus  and  an  organism  incapable  of  causing  "  retting  "  by 
itself.  The  organisms  discovered  by  Prazmowski  and  Pasteur  were 
anaerobic.  But  Fitz  and  Hueppe  isolated  an  aerobic  butyric  bacillus. 
This  fact  was  confirmed  by  Gruber,  working  with  a  pure  culture  in 
1887,  and  it  was  at  the  same  time  demonstrated  that  the  Clostridium 
lutyricum  of  Prazmowski  consists  of  a  number  of  closely  allied,  but 
distinct,  species.  Lafar  states  that  nearly  related  to  this  is  a  ferment 
isolated  by  Liborius  from  old  cheese,  and  introduced  into  literature 
under  the  name  of  Clostridium  fcetidum.  This  organism  liberates 
foul-smelling  gases,  in  addition  to  producing  butyric  acid,  and  forms 
one  of  the  many  connecting  links  between  the  butyric  acid  bacteria 
and  the  so-called  "  potato  "  bacilli.  No  sharply  defined  limit  can  be 
drawn  between  these  two  groups. 

The  following  are  the  three  chief  organisms  of  butyric  acid : — 

1.  Bacillus  Butyricus  (Botkin) 

Source  and  habitat— Widely  distributed  in  milk,  water,  soil,  dust. 

Morphology— Hods,  1  to  3  ^  long,  0'5  ^  thick.  Sometimes  in  threads,  sometimes 
in  chains. 

Staining  reaction— Stains  by  Gram's  method. 

Motility— Motile. 

Spore  formation — Spores  in  middle  or  at  end  of  bacillus  ;  about  1  /x  thick 
(provisional)  ;  sporulation  not  proved  (Botkin). 

Biology :  cultural  characters  (including  biochemical  features')— Favourable  tempera- 
ture 37°  C.  ;  organism  contains  starch  granules. 

Bouillon — Slight  growth ;  at  18°  C.  involution  forms  may  occur.  Vigorous 
growth  if  glucose  present,  with  opaque  turbidity. 

Gelatine  plates  and  tubes — Round  or  oval  colonies  with  sinuous  edges  ;  medium 


BUTYRIC  ACID  FERMENTATION  109 

is  rapidly  liquefied  ;  gas  development ;  slight  undulating  colonies,  as  if  consisting  of 
mass  of  felted  threads.  No  odour. 

Agar  plates  and  tubes — A  luxuriant  growth  with  gas  development  and  ramifica- 
tions in  medium.  Odour  present. 

Potato — Growth  extends  into  potato  substance ;  smell  of  alcohol. 

Milk— At  37°  C.,  after  fifteen  hours  casein  precipitated  and  butyric  acid  is  formed 
without  intermediate  formation  of  lactic  acid.  Coagulum  eventually  dissolves  ; 
before  that  stage  the  appearance  is  very  characteristic ;  there  is  a  spongy  fatty 
layer  on  surface,  then  clear  fluid,  and  then  a  white  deposit  The  presence  of  this 
organism  is  readily  proved  in  almost  all  milk.  Fill  a  half  litre  flask  with  milk,  and 
steam  at  100°  C.  for  half  an  hour.  Incubate  at  37°  C.  In  less  than  twenty-four 
hours  the  characteristic  changes  will  occur,  with  strong  odour  of  butyric  acid. 
Care  must  be  taken  that  the  gas  pressure  does  not  burst  the  flask.  There  is  a 
marked  odour  of  butyric  acid.  Other  acids  present  are  acetic,  formic,  and  lactic. 

Anaerobic  or  aerobic — Facultative  anaerobe. 

Non-pathogenic — It  has  been  suggested  that  the  B.  enteritidis  sporogtnes  of  Klein 
is  a  pathogenic  form  of  this  bacillus. 

2.  Bacillus  Butyricus  (Hiippe) 

Source  and  habitat — Milk. 

Morphology —Slender  rods  ;  1-2  to  4  /A  long,  0'5  //.  thick;  round  ends.  May 
grow  into  filaments  ;  rods  slightly  bent ;  21  n  long,  0'3  A*  broad. 

Staining  reaction — Stains  by  Gram's  method. 

Flagella  ;  motility— Many  flagella  ;  actively  motile. 

Spore  formation — Oval  spores  at  37°  C.  ;  mesially  situated. 

Biology :  cultural  characters  (including  biochemical  features'). 

Bouillon— A  pellicle  is  formed  ;  bouillon  remains  clear.     No  indol. 

Gelatine  plates  and  tubes  —  Small  whitish-yellow  colonies  with  crater-shaped 
depression ;  liquefaction ;  whitish-grey  wrinkled  pellicle  produced  in  liquid 
cultures  in  tubes  ;  liquefied  medium  cloudy  and  yellowish  in  colour. 

Agar  plates  and  tubes — A  thin  yellow  layer,  similar  to  B.  mesentericus. 

Potato — A  fawn-coloured  transparent  layer,  sometimes  wrinkled.  Somewhat 
similar  to  B.  megatherium. 

Milk — Is  coagulated.  Precipitated  casein  subsequently  dissolved.  Bitter  taste. 
Butyric  acid  produced  from  salts  of  lactic  acid  ;  also  from  milk-sugar  when  it  is 
previously  hydrated. 

Facultative  anaerobe. 

Non-pathogenic. 

3.  Bacillus  Butyricus  (Pasteur).     (Vibrion  Butyrique) 

Source  and  habitat— MY,  and  thence  to  milk. 

Morphology — Cylindrical  rods  with  rounded  extremities  5  3  /*  to  5  /u  long  by 
•6  fi  to  '8  /i  broad.  Isolated  or  in  chains  ;  at  times  in  long  filaments  indistinctly 
articulated. 

Staining  reaction — Ordinary  aniline  stains. 

Motility— Feebly  motile ;  motility  ceases  at  once  in  presence  of  free  oxygen. 

Spore  formation — Ovoid  spores. 

Biology  :  cultural  characters  (including  biochemical  features'). 

Bouillon — Grows  freely  under  strictly  anaerobic  conditions  in  bouillon  contain- 
ing lactate  of  lime. 

Agar  plates  and  tubes — In  agar  "  shake  "  cultures  free  from  oxygen  the  medium 
becomes  clouded  in  the  lower  portion,  and  is  soon  broken  up,  with  copious  gas 
formation  accompanied  by  strong  smell  of  butyric  acid. 

Gelatine  plates  and  tubes — As  upon  agar,  but  in  a  less  degree,  the  medium 
liquefying  in  the  neighbourhood  of  the  forming  colonies. 

Anaerobic. 

Non-pathogenic. 

Several  other  butyric  acid  organisms  have  been  isolated,  of  which  a  few  notes 
may  be  added  : — 


110  BACTERIA  AND  FERMENTATION 

Bacillus  acidi  butyrici— (Kedrowski's  Butyric  acid  bacillus).  Anaerobic. 
Kedrowski  (Z.  16.  3)  has  isolated  from  mixtures  of  sugar  solution  with  bad  cheese 
or  rancid  cream-butter  which  has  been  placed  in  the  incubator,  two  organisms 
which  only  show  small  deviations  from  one  another.  (Gf.  the  B.  saccharo-bulyricus 
of  Klencki  from  cheese).  Kedrowski's  B.  acidi  butyrici  is  a  motile  bacillus,  which 
towards  one  end  produces  ellipsoidal  spores.  The  staining  of  the  spores  is  readily 
accomplished  The  colonies  in  gelatine  show  rays — those  in  agar  partly  reticulated — 
and  interlaced  spurs.  Liquefaction  of  gelatine  is  more  or  less  marked.  Milk  is 
coagulated  with  separation  of  serum  on  the  surface  (acid  reaction).  There  is 
gradual  peptonisation  and  simultaneous  gas  development. 

Although,  according  to  Pasteur's  researches,  the  butyric  acid 
ferment  performs  its  function  anaerobically,  many  butyric  organisms 
can  act  in  the  presence  of  oxygen,  and  yield  somewhat  different 
products. 

All  of  them,  however,  ferment  most  actively  at  a  temperature  at 
or  about  blood-heat,  and  the  spores  are  able  to  withstand  boiling  for 
from  three  to  twenty  minutes  (Fitz).  It  will  be  observed  that  as  in 
lactic  acid  fermentation  so  in  butyric,  the  results  are  not  due  to  one 
species  only. 

5.  Ammoniacal  Fermentation  (see  under  Soil). 

From  what  has  now  been  said,  it  is  obvious  that  although  we  learn 
many  important  facts  by  a  study  of  these  different  forms  of  fermen- 
tation, we  may  also  learn  on  the  one  hand  how  to  prevent  or  correct 
those  conditions  constantly  occurring  in  fermented  beverages,  which 
are  known  as  "  diseases,"  and  on  the  other,  the  opportunities  which 
occur  in  industrial  processes  for  the  application  of  fermentation. 
We  will  first  deal  with  the  former. 


Diseases  in  Beer  and  Wines 

We  have  seen  how  the  knowledge  of  fermentation  has  been  com- 
piled by  a  large  number  of  workers.  Spallanzani,  Schwann,  Pasteur, 
and  Hansen  all  contributed  epoch-making  researches.  In  the  same 
way  the  investigations  of  diseases  in  beers  and  wines  were  carried  on 
by  many  observers,  and  were,  at  all  events  in  the  early  stage,  closely 
connected  with  those  relating  to  spontaneous  generation  and  mixed 
cultures  of  bacteria,  or  of  yeasts  occurring  in  fermentation.  These 
so-called  "diseases"  are  analogous  to  the  taints  occurring  in  milk. 
It  was  in  1883  that  Hansen  demonstrated  that  the  universally 
dreaded  yeast  turbidity  and  the  disagreeable  changes  in  taste,  odour, 
or  colour  of  beer  were  caused  not  by  the  water  or  malt  or  particular 
method  of  brewing,  as  was  commonly  believed,  but  that  these 
diseases  had  their  origin  in  micro-organisms  or  in  the  yeast  itself.* 
A  clue  had  been  given  by  Scheele  and  Appert,  who  had  prevented 
these  diseased  conditions  by  physical  agents  which  had  destroyed  the 

*  Practical  Studies  in  Fermentation,  E.  C.  Hansen,  pp.  156-231. 


DISEASES  OF  BEER  111 

organisms  able  to  produce  the  diseases.  The  demonstration  by 
experiment  of  the  cause  of  these  diseases  was  worked  out  by  Pasteur, 
who,  as  we  have  seen,  established  the  fact  that  there  are  different 
micro-organisms  inducing  different  kinds  of  fermentation,  and  there- 
fore if  it  be  desired  to  procure  a  pure  fermentation,  a  pure  and 
not  a  mixed  ferment  must  be  used  at  the  commencement;  and 
immediately  after  the  primary  fermentation  the  wine  must  be 
"  pasteurised  "  to  destroy  the  disease-producing  organisms.  In  short, 
disease-producing  organisms  must  either  be  excluded  from,  or  killed 
in,  the  wine. 

By  carrying  out  a  large  number  of  experiments,  partly  with 
single  species  of  yeast,  and  partly  with  mixtures,  Hansen  was  able  to 
declare  that  many  of  these  diseases  were  due  to  particular  yeasts. 
The  number  of  such  yeasts  is  by  no  means  small.  Hence  we  have 
two  groups  of  yeasts,  namely,  "  culture  "  or  "  brewery  yeasts,"  those 
that  are  employed  in  brewing ;  and  "  wild  yeasts,"  occurring  widely 
distributed  in  nature,  and  which  on  gaining  entrance  to  breweries  set 
up  diseases  in  the  fermentations.  The  development  of  wild  yeasts  is 
promoted  by  vigorous  aeration  of  the  beer  whilst  it  is  being  drawn 
off,  and  also  through  the  bottles  being  badly  corked.  Beer  which  has 
undergone  a  feeble  fermentation,  and  which  has  a  high  extract,  is 
more  subject  to  contamination  than  a  beer  which  has  not.  When 
beer  which  has  remained  sound  in  the  larger  casks  is  attacked  after 
it  has  been  drawn  off,  it  is  clear  that  the  agent  of  the  disease 
obtained  entrance  into  the  beer  from  the  surrounding  air  or  from 
unclean  vessels.  If  the  infection  is  not  great  in  amount,  it  may, 
particularly  in  a  good  beer,  have  practically  no  effect.  There  can  be 
no  doubt  that  some  of  the  Saccharomycetes  can  live  for  months  in  soil 
and  dust,  even  atmospheric  dust,  and  amongst  these  may  be  various 
disease-yeasts. 

The  diseases  of  wines  and  beers  are  various.  Generally  speaking, 
the  chief  forms  are  comprised  in  the  following  simple  classifica- 
tion : — 

1.  Turbidities. — (a)  Gluten  turbidities,  or  albuminous  scud,  due 
to  precipitation  of  albuminoids. 

(b)  Chemical   suspension    and    deposits,   e.g.    calcium    tartrate, 
reduced  sulphur  scud,  resins,  essential  oils,  etc. 

(c)  Starch  turbidity,  due  to  the  presence  of  unsaccharified  starch. 

(d)  Yeast  turbidity,  due  to  a  high  content  of  yeast  cells. 

(e)  Bacterial  turbidity,  brought  about  by  fission  fungi. 

2.  Ropiness,  which  may  be  thus  classified  separately,  although 
doubtless  frequently  due  to  a  high  degree  of  turbidity.     This  con- 
dition of  ropiness  in  wine,  formerly  attributed  to  a  coagulation  of  the 
albuminoids,   was   traced   by   Pasteur    to   a    number   of   organisms 
of  which  he  described  two  chief  forms,  namely,  a  streptococcus  and 


112  BACTERIA  AND  FERMENTATION 

Bacillus  viscosus  vini.  This  latter  organism  occurs  in  the  form  of 
small  rods,  frequently  united  in  pairs,  and  capable  of  producing 
ropiness  in  white  wines  in  the  absence  of  air.  The  presence  of  sugar 
is  a  sine  qud  non  for  the  occurrence  of  the  malady,  since  it  forms  the 
material  from  which  the  strings  of  mucus  are  produced.  Nessler 
maintains  that  wines  containing  over  10  per  cent,  of  alcohol  are  proof 
against  ropiness. 

Pasteur  also  investigated  ropiness  in  beer,  and  traced  it  to 
Micrococcus  mscosus.  But  in  all  probability  there  are  a  number  of  the 
Schizomycetes  possessing  the  power  of  rendering  beer  and  wine  viscid. 
The  so-called  Sarcina  turbidity  of  beer  has  been  traced  to  the  Pedio- 
coccus  cerevisice.  But  it  should  be  borne  in  mind  that  such  conditions 
may  be  easily  mistaken  for  turbidities  set  up  in  other  ways. 

3.  Changes  in  Colour. — The  browning  of   wines — changing   of 
colour  with  turbidity  and  unpleasant  flavour,  sometimes  occurring  in 
white  wines — is  said  to  be  due  to  oxydaxes,  enzymes  produced  by 
some  of  the  yeasts  and  setting  up  an  oxidation. 

4.  Alteration  of  Flavour,  Bitterness,  Acidity,  etc.— Bitterness 
of  wine  almost  exclusively  affects  red  wines.     The  wine  decolorises 
and  develops  a  strange  odour   and   a   bitter   after-taste.      Pasteur 
attributed  the  disease  to  bacteria,  but  up  to  the  present  no  species 
has  been  isolated  able  to  bring  about  this  condition  upon  inoculation 
in  healthy  wines.     Bittering  of  beer  may  be  occasioned  by  a  disease- 
yeast  (Saccharomyces  pastorianus  /.)  introduced  at  the  commencement 
of  the  primary  fermentation,  even  in  such  small  quantity  as  one-fifth 
of  the  pitching  yeast.     This  organism,  according  to  Hansen,  not  only 
injuriously   affects   the   taste   and  odour  of   the  beer,  but  also  its 
stability.     It  is  of  very  frequent  occurrence  in  breweries. 

The  turning  (tourne)  of  wines  is  by  no  means  a  clearly-defined  or 
uniform  phenomenon.  The  most  frequent  form,  perhaps,  is  that  due 
to  the  vinegar  taint  (caused  by  Mycoderma  aceti).  But  the  condition 
may  be  set  up  by  the  lactic  acid  bacteria.  It  mostly  attacks  young 
vintages.  The  wine  becomes  turbid,  eventually  having  an  appearance 
of  diluted  milk,  and  even  later  it  may  assume  a  condition  of  brown 
or  inky-black  liquid. 

The  turning  of  beer,  on  the  other  hand,  is  a  simple  malady  due 
to  lactic  acid  fermentation,  set  up  by  the  Saccharo-bacillus  pastorianus 
III.  The  beer  at  first  loses  its  brightness,  then  becomes  turbid,  and 
ultimately,  according  to  some  authorities,  of  unpleasant  smell  and 
taste.  If  the  sample  be  shaken  delicate  waves  or  films  of  the  organ- 
ism are  apparent  to  the  naked  eye,  and  eventually  the  beer  becomes 
muddy.  Hansen  has  shown  that  there  are  two  species  of  yeast,  S. 
pastor.  III.  and  S.  ellipsoideus  II.,  which  produce  the  disease  when 
they  are  present  in  the  pitching  yeast,  and  are,  therefore,  introduced  at 
the  commencement  of  the  primary  fermentation.  Both  species  are 


INDUSTRIAL  APPLICATIONS  113 

injurious  when  present  at  this  stage,  and  indeed  only  at  this  stage. 
S.  ellipsoideus  II.  is  the  stronger  of  the  two  species.  Whilst  upon  this 
particular  subject,  we  may  add  that  in  1883  Hansen  demonstrated 
that  these  much-dreaded  turbidities  and  other  beer  diseases  may  be 
due  to  mixtures  of  two  yeasts,  even  though  each  of  them  by  itself 
gives  a  faultless  product. 

The  Industrial  Application  of  Bacterial  Ferments 

We  may  commence  our  brief  category  of  the  industrial  application 
of  bacteria  by  referring  the  reader  to  fermentations,  like  the  acetous 
(which  results  in  the  manufacture  of  vinegar),  the  alcoholic  (alcoholic 
beverages),  the  lactic  acid  (souring  of  milk  for  dairying  purposes, 
cheese,  etc.),  the  butyric  (resulting  in  butyric  acid),  and  those  fer- 
mentations occurring  in  the  soil  and  improving  the  fertility  of  land 
for  farming  purposes.  With  the  principal  facts  concerning  each  of 
these  applications  of  bacteria  to  industrial  processes  we  have  dealt 
elsewhere.  It  remains  for  us  to  mention  other  spheres  of  industry 
where  bacteria  are,  whether  we  recognise  it  or  not,  playing  a  leading 
rolfj.  Their  industrial  effects  are  often  secondary  to  vital  processes. 
For  instance,  in  securing  their  food  bacteria  break  down  organic 
material  and  bring  about  chemical  and  physical  change.  Now  this 
power  which  organisms  have  of  chemically  destroying  compounds 
may,  or  may  not,  be  of  primary  importance,  but  there  can  be  no  doubt 
that  many  of  the  products  which  arise  as  a  result  are  of  an  importance 
in  the  world  which  it  is  difficult  to  over-estimate.  Perhaps  the  most 
remarkable  examples  occur  in  soil  and  in  milk.  But  other  illustra- 
tions which  will  at  once  occur  to  the  reader  are  the  maceration 
industries.  For  example,  linen,  as  is  well  known,  is  produced  from 
flax.  The  flax  stem  is  made  up  of  cellular  substance,  flax  fibres  and 
wood  fibres ;  the  latter  are  of  no  service  in  the  making  of  linen,  but 
the  whole  is  bound  together  by  a  gummy,  resinous  substance  termed 
"the  central  lamellae"  (an  intermediate  inter-cellular  substance 
consisting  probably  not  of  pectose,  but  of  calcium  pectate).  The 
solution  of  this  cementing  substance  can  be  brought  about  by 
chemical  means  by  treating  the  plant  with  very  dilute  sulphuric 
acid  and  then  neutralising  the  adherent  acid  by  a  weak  alkali  bath 
(Bura).  But  it  can  also  be  solved  by  the  process  known  as  retting. 
There  is  dew-retting  and  water-retting.  The  former  is  practised  in 
Russia,  and  consists  in  spreading  the  flax  on  the  grass  and  exposing 
it  to  the  influence  of  clew,  air,  rain,  and  light.  The  result  is  a  soft 
and  silky  fibre.  Water-retting  is  the  method  more  commonly 
adopted,  and  is  accomplished  by  means  of  steeping  the  flax  in  bundles, 
roots  downwards,  in  tanks  or  ponds,  with  appliances  so  arranged  as 
to  keep  the  flax  below  water.  In  ten  to  fourteen  days,  according  to 

H 


114  BACTERIA  AND  FERMENTATION 

the  warmth  of  the  weather,  fermentation  is  completed  by  the  break- 
ing away  of  the  "  shore  "  or  "  shive  "  (the  woody  core)  from  the  flax 
fibres.  This  decomposition  and  eventual  breaking-down  is  due  to 
bacteria,  which,  under  favourable  circumstances,  multiply  rapidly 
and  set  up  the  decomposition  of  the  pectin  resinous  substance. 
Winogradsky,  in  1895,  proved  that  the  process  was  due  to  a  large 
bacillus  (10-15  //,  long,  1  //,  broad).  It  is  an  anaerobe,  growing  not 
in  gelatine,  but  in  the  presence  of  nitrogenous  food  will  ferment 
saccharose,  lactose,  and  starch. 

A  precisely  similar  process  is  used  in  the  preparation  of  jute  and 
hemp.  The  former  is  of  course  used  in  various  fabric  industries,  the 
chief  centre  of  such  manufactures  being  at  Dundee.  Jute  fibre  is 
obtained  from  the  bark  of  at  least  two  species  of  plants  allied  to  the 
lime-tree  order.  The  fibre,  which  is  the  inner  bark,  is  separated  from 
the  stem  by  retting,  either  in  rivers  or  tanks.  The  retting  lasts  for 
different  periods,  from  two  days  to  three  weeks,  and  when  the 
cementing  substance  between  the  fibres  and  the  stem  is  sufficiently 
decomposed  to  allow  of  it,  the  jute  fibre  is  separated,  and  may  be 
woven  into  sacking,  carpets,  curtains,  etc.  It  is  said  that  many  of 
the  brightly-dyed  prayer-carpets  used  in  the  East  by  Moslems  are 
made  of  this  material  in  Dundee,  and  exported.  Hemp  also  is 
cultivated  in  Poland  and  European  Russia  for  the  sake  of  its  fibre, 
which  is  used  for  sail-cloth  and  other  coarse  material.  This  fibre  is 
also  separated  by  retting.  Another  example  of  the  same  putrefactive 
process  is  the  preparation  of  cocoanut  fibre  for  matting,  etc.  Some- 
times retting  for  as  long  as  twelve  months  is  necessary  to  separate 
the  fibres  from  the  unripe  husk  of  the  cocoanut.  Sponges  are  cleared 
in  much  the  same  manner  by  the  putrefaction  and  softening  of  the 
organic  matter  in  their  interstices,  set  up  by  micro-organisms.  The 
preparation  of  indigo  from  the  indigo  plant  is  brought  about  by  a 
special  bacterium  found  on  the  leaves.  If  the  leaves  are  sterilised 
no  fermentation  occurs,  and  no  indigo  is  formed.  If,  however,  some 
of  the  specific  bacteria  are  added  to  the  mass,  the  fermentation  soon 
begins,  and  the  blue  colour  of  the  indigo  makes  its  appearance.  In 
the  treatment  of  ox-hides  for  the  production  of  certain  kinds  of 
leather  the  first  object  of  the  tanner  is  to  clean  and  soften  the  hide, 
which  is  accomplished  by  washing.  The  unhairing  and  removal  of 
the  scarf-skin  is  the  next  operation,  and  this  is  achieved  in  America 
by  "  sweating "  the  hides,  or  artificially  heating  them  till  incipient 
putrefactive  fermentation  is  set  up  by  means  of  bacteria.  Even  in 
the  subsequent  tanning  bacteria  probably  play  an  important  part. 
But  little  is  known  at  present  of  their  work  in  this  respect. 

In  the  production  of  tobacco,  the  leaves,  when  gathered,  are  allowed 
to  become  somewhat  withered,  and  are  then  arranged  in  moderate- 
sized  heaps,  where  they  undergo  a  so-called  "  sweating,"  after  which 


INDUSTRIAL  APPLICATIONS  115 

they  are  tied  in  bundles  and  arranged  in  huge  heaps,  containing 
sometimes  50  tons  of  tobacco.  Hereupon  active  decomposition 
rapidly  ensues,  and  the  temperature  rises  to  50°  or  60°  C.  This 
fermentation  is  due  to  bacteria,  and  was  studied  by  Schloesing  and 
Suchslan,  who  used  pure  cultures  of  bacteria  for  the  purpose  of 
favourably  influencing  the  fermentation  of  tobacco,  and  producing 
a  definite  aroma.  There  is  some  evidence  to  show  that  certain  of 
the  family  of  Aspergillus  co-operate  with  the  bacteria  in  this  process. 
Throughout  the  needful  operations  in  tobacco-curing  the  producer 
has  to  contend  with  a  number  of  micro-organisms  which  may  produce 
disease  in  the  tobacco. 

The  fermentation  of  cellulose  is  an  example  of  bacterial  action 
which  has  been  more  or  less  widely  applied  to  industry.  The 
process  is  due  to  Bacillus  amylobacter,  which  acts,  it  is  supposed,  in 
symbiotic  relationship  with  some  other  organism  incapable  of 
fermenting  cellulose  by  itself.  In  relation  to  these  so-called 
industrial  symbioses  it  will  be  remembered  by  some  that  Calmette 
drew  attention  at  the  British  Association  Meeting  at  Dover  (1899) 
to  the  application  of  bacteria  to  various  processes  carried  out  in  the 
East.  For  example,  the  Japanese  manufacture  their  scike  with  a 
form  of  aspergillus  described  by  Ahlburg  in  1879,  and  the  cau  de  vie 
and  vins  de  riz  of  the  Chinese  and  Javanese  have  their  source  in 
symbiotic  fermentations.  Thus,  in  many  cases,  without  the  manu- 
facturer even  knowing  it,  micro-organic  ferments  are  utilised  in 
industrial  operations. 

In  all  these  applications  it  is  obvious  we  have  advanced  only  the 
first  stage  of  the  journey.  Nevertheless,  here,  as  in  nature  on  a 
large  scale  in  the  formation  of  fertile  soils  and  coal  measures,  we  find 
bacteria  or  their  allies  silently  at  work  achieving  great  ends  by 
co-operating  in  countless  hordes. 


CHAPTER  V 


BACTERIA  IN  THE  SOIL 

Methods  of  Examination — Methods  of  Anaerobic  Culture — Place  and  Function  of 
Micro-organisms  in  Soil  —  Denitrification,  Nitrification,  Nitrogen-fixation, 
Bacterial  Symbiosis  —  Saprophytic  and  Pathogenic  Organisms  in  Soil  — 
Tetanus  —  Quarter  -  Evil  —  Malignant  CEdema  —  The  Relation  of  Soil  to 
Bacterial  Diseases,  such  as  Typhoid  Fever. 

SURFACE  soils  and  those  rich  in  organic  matter  supply  a  varied  field 
for  the  bacteriologist.  Indeed,  it  may  be  said  that  the  introduction 
of  the  plate  method  of  culture  and  the  improved  facilities  for 
growing  anaerobic  micro-organisms  have  opened  up  possibilities  of 
research  into  soil  micro-biology  unknown  to  previous  generations  of 
workers. 

From  the  nature  of  bacteria  it  will  be  readily  understood  that 
their  presence  is  affected  by  physical  conditions  of  the  soil,  and  in 
all  soils  they  occur  only  within  a  few  feet  of  the  surface.  As  we  go 
down  below  2  feet,  bacteria  become  less,  and  below  a  depth  of 
5  or  6  feet  we  only  find  a  few  anaerobes.  At  a  depth  of  10  feet, 
and  in  the  "ground  water  region,"  bacteria  are  scarce  or  absent. 
This  is  held  to  be  due  to  the  porosity  of  the  soil  acting  as  a  filtering 
medium.  Eegarding  the  numbers  of  micro-organisms  present  in  soil, 
no  very  accurate  standard  can  be  obtained.  Ordinary  earth  may 
yield  anything  from  10,000  to  5,000,000  per  gram,  whilst  from 
polluted  soil  even  100,000,000  per  gram  have  been  estimated. 
These  figures  are  obviously  only  approximate,  nor  is  an  exact 
standard  of  any  great  value.  Nevertheless  Frtinkel,  Beumer,  Miquel, 
and  Maggiora  have,  as  the  result  of  experiments,  arrived  at  a  number 
of  conclusions  respecting  bacteria  in  soil  which  are  of  practical  use. 
From  these  results  it  appears  that,  in  addition  to  the  "  ground  water 
region  "  being  free,  or  nearly  so,  virgin  soils  contain  much  fewer  than 
cultivated  lands,  and  these  latter,  again,  fewer  than  made  soils  and 

116 


PLATE  9. 


METHODS  OF  EXAMINATION  117 

inhabited  localities.  In  cultivated  lands  the  number  of  organisms 
augments  with  the  activity  of  cultivation  and  the  strength  of  the 
fertilisers  used.  In  all  soils  the  maximum  occurs  in  July  and 
August. 

But  the  condition  which  more  than  all  others  controls  the  quantity 
and  quality  of  the  contained  bacteria  is  the  degree  and  quality  of 
the  organic  matter  in  the  soil.  The  quantity  of  organic  matter 
present  in  soil  having  a  direct  effect  upon  bacteria  will  be  materially 
increased  by  placing  in  soil  the  bodies  of  men  and  animals  after 
death.  Dr  Buchanan  Young  two  or  three  years  ago  performed  some 
experiments  to  discover  to  what  degree  the  soil  bacteria  were  affected 
by  these  means.  "  The  number  of  micro-organisms  present  in  soil 
which  has  been  used  for  burial  purposes,"  he  concludes,  "exceeds 
that  present  in  undisturbed  soil  at  similar  level,  and  that  this  excess, 
though  apparent  at  all  depths,  is  most  marked  in  the  lower  reaches 
of  the  soil."*  The  numbers  were  as  follows  : — 

Virgin  soil,  4  ft.  6  in.    =  53,436  m.o.  per  gram  of  soil. 
Burial  soil  (8  years),  4  ft.  6  in.    =   363,411  m.o.  per  gram  of  soil. 
(3     „    ),  6  ft.  6  in.   =   722,751 

Methods  of  Examination  of  Soil.— Two  simple  methods  are  generally  adopted. 
The  first  is  to  obtain  a  qualitative  estimation  of  the  organisms  contained  in  the  soil. 
It  consists  simply  in  adding  to  test-tubes  of  liquefied  gelatine  or  broth  a  small 
quantity  of  the  sample,  finely  broken  up  with  a  sterile  rod.  The  test-tubes  are  now 
incubated  at  37°  C.  and  22°  C.,  and  the  growth  of  the  contained  bacteria  observed 
in  the  test-tube,  or  after  a  plate  culture  has  been  made  on  gelatine,  agar,  or  glucose- 
litmus  agar.  The  second  plan  is  adopted  in  order  to  secure  more  accurate  quanti- 
tative results.  One  gram  or  half-gram  of  the  sample  is  weighed  on  the  balance, 
and  then  added  to  100  c.c.  or  1000  c.c.  of  distilled  sterilised  water  in  a  sterilised 
flask,  in  which  it  is  thoroughly  mixed  and  washed.  From  either  of  these  two 
different  sources  it  is  now  possible  to  make  sub-cultures  and  plate  cultures.  The 
procedure  is,  of  course,  that  described  under  the  examination  of  water  (p.  463  at  seq.), 
and  Petri's  dishes,  Koch's  plates,  or  Esmarch's  roll  cultures  are  used.t  Many  of 
the  commoner  bacteria  in  soil  will  thus  be  detected  and  cultivated.  Spores  may 
be  isolated,  as  is  described  under  Examination  of  Sewage.  But  it  is  obvious  that  this 
by  no  means  covers  the  required  ground.  It  will  be  necessary  for  us  here  to  con- 
sider the  methods  generally  adopted  for  growing  anaerobic  bacteria,  that  is  to  say, 
those  species  which  will  not  grow  in  the  presence  of  oxygen.  This  anaerobic 
difficulty  may  be  overcome  in  a  variety  of  ways. 


Methods  of  Anagrobic  Cultivation 

1.  The  oxygen  may  be  displaced  by  some  other  gas,  and  though  coal-gas, 
nitrogen,  and  carbon  dioxide  may  all  be  used  for  this  purpose,  it  has  become  the 
almost  universal  practice  to  grow  anaerobes  in  hydrogen.  The  hydrogen  is  readily 
obtained  by  Kipp's  or  some  other  suitable  apparatus  for  the  generation  of  hydrogen 


*  Proc.  Royal  Soc.  of  E din.,  xxxvii.,  pt.  iv.,  p.  759. 

t  See  also  Report  of  the  Medical. Officer  to  the  Local  Government  Board  (1897-98), 
A.  C.  Houston,  pp.  251-307. 


118 


BACTERIA  IN  THE  SOIL 


from  zinc  and  dilute  sulphuric  acid,  or  it  may  be  provided  in  a  cylinder.  The  free 
gas  is  passed  through  various  washbottles  to  purify  it  of  any  contaminations ;  e.g. 
lead  acetate  (1-10  of  water)  removes  any  traces  of  sulphuretted  hydrogen,  silver 
nitrate  (1-10)  doing  the  same  for  arseniated  hydrogen  ;  whilst  a  flask  of  pyrogallate 
of  potash  will  remove  any  oxygen.  It  is  not  necessary  to  have  these  three  purifiers 
if  the  zinc  used  in  the  Kipp's  apparatus  is  pure.  Occasionally  a  fourth  flask  is  added 
of  distilled  water,  and  this,  or  a  dry  cotton-wool  stopper  in  the  exit  tube,  will  ensure 
germ-free  gas.  From  the  further  end  of  the  exit  tube  of  the  Kipp's  apparatus  an 
indiarubber  tube  will  carry  the  hydrogen  to  its  desired  destination.  With  some  it  is 
the  custom  to  place  anaerobic  cultures  in  test-tubes,  and  the  test-tubes  in  a  large 
flask,  tube,  or  desiccator,  having  a  two-way  tube  for  entrance  and  exit  of  the 
hydrogen,  or  Petri  dishes  may  be  used  and  placed  in  well-sealed  jar  or  desiccator  ; 
others  prefer  to  pass  the  hydrogen  immediately  into  a  large  test-tube  containing  the 
culture  (Frankel's  method).  Either  method,  if  properly  carried  out,  will  be  found 
effectual,  and  the  growth  of  the  culture  in  hydrogen  is  readily  observed.  Another 
plan  is  to  use  a  yeast  flask,  and  after  having  passed  the 
hydrogen  through  for  about  half  an  hour,  the  lateral  exit 
tube  is  dipped  into  a  small  capsule  containing  mercury 
(as  in  Plate  9).  The  entrance  tube  is  now  sealed,  and 
the  whole  apparatus  placed  in  the  incubator.  The 
interior  of  the  flask  containing  the  culture  is  filled  with 
an  atmosphere  of  hydrogen.  No  oxygen  can  obtain 
entrance  through  the  sealed  entrance  tube,  or  through 
the  exit  tube  immersed  in  mercury  Yet  through  this 
latter  channel  any  gases  produced"  by  the  culture  may 
escape. 

2.  The  Absorption  Method. — Instead  of  adding  hydro- 
gen to  the  tube  or  flask  containing  the  anaerobic  culture, 
it  is  feasible  to  add  to  the  medium  substances,  such  as 
glucose  or  pyrogallic  acid,  which  will  absorb  the  oxygen 
which  is  present,  and  thus  enable  the  anaerobic  require- 
ment to  be  fulfilled.  To  various  media — gelatine,  agar, 
or  broth  (the  latter  used  for  obtaining  the  toxins  of 
anaerobes) — 2  per  cent,  of  glucose  may  be  added. 
Pyrogallic  acid,  or  pyrogallic  acid  one  part  and  20  per 
cent,  caustic  potash  one  part,  is  also  readily  used  for 
absorptive  purposes.  A  large  glass  tube  of  25  c.c.  height, 
termed  a  Buchner's  cylinder,  having  a  constriction  near 
the  bottom,  is  taken  ;  and  about  two  drachms  of  the 

FIG.  is.— FBANKKL'S  TUBE.      pyrogallic  solution  are  placed  in  the  bulb.     A  test-tube 
For  Cultivation  of  Anaerobes,    containing  the  culture  is  now  lodged  in  the  upper  part 
above  the  constriction,  and  the  mouth  of  the  Buchner 

tube  is  carefully  sealed.  The  apparatus  is  then  placed  in  the  incubator  at  the  desired 
temperature,  and  the  contained  culture  grows  under  anaerobic  conditions.  As  the 
pyrogallic  solution  absorbs  the  oxygen  it  assumes  a  darker  tint. 

3.  Mechanical  Methods.  — These  include  various  ingenious  methods  for  preventing 
an  admittance  of  oxygen  to  the  culture.     An  old-fashioned  one  was  to  plate  out  the 
culture  and  protect  it  from  the  air  by  covering  it  with  a  plate  of  mica.     A  more 
serviceable  mode  is  to  inoculate,  say,  a  tube  of  agar  with  the  anaerobic  organism, 
and  then  pour  over  the  culture  a  small  quantity  of  melted  agar,  which  will  readily 
set,  and  so  protect  the  culture  itself  from  the  air.     Oil  or  vaseline  may  be  used 
instead  of  melted  agar.     Another  mechanical  method  is  to  make  a  deep  inoculation, 
and  then  melt  the  top  of  the  medium  over  a  Bunsen  burner,  and  thus  close  the 
entrance  puncture  and  seal  it  from  the  air. 

4.  Absorption  of  Oxygen  by  an  Aerobic  Culture. — This  method  takes  advantage 
of  the  power  of  absorption  of  certain  aerobic  bacteria,  which  are  planted  over  the 
culture  of  the  anaerobic  species.     It  is  not  practically  satisfactory,  though  occasionally 
good  results  have  been  obtained. 

5.  Lastly,  there  is  the  Vacuum  Method. — By  this  means  it  is  obviously  intended 
to  extract  air  from  the  culture  and  seal  it  in  vacuo.     The  culture  tubes  are  connected 


PLATE  10. 


A  VACUUM  METHOD  OF  ANAEROBIC  CULTURE. 


[To  face  page  US. 


"    OF  T*€ 

UNIVERSITY    ) 


OF 


QUALITATIVE  EXAMINATION  119 

with  the  air-pump,  and  exhausted  as  much  as  possible.  The  method  can  be  applied 
in  many  different  ways  (for  example,  with  pyrogallic  solution,  as  in  Bulloch's 
apparatus). 

Of  these  various  methods  it  is  on  the  whole  best  to  choose  either  the  hydrogen 
method,  the  vacuum,  or  the  plan  of  absorption  by  grape-sugar  or  pyrogallic.  In 
anaerobic  plate  cultures  grape-sugar  agar  plus  0'5  per  cent,  of  formate  of  soda  may 
be  used.  The  poured  inoculated  plate  should  be  placed  over  pyrogallic  solution 
under  a  sealed  bell-glass  and  incubated  at  37°  C.  Pasteur,  Roux,  Joubert,  Chamber- 
land,  Esmarc-h,  Kitasato,  and  others  have  introduced  special  apparatus  to  facilitate 
anaerobic  cultivation,  but  the  principles  adopted  are  those  which  have  been 
mentioned. 

The  Qualitative  Examination  of  Bacteria  in  the  Soil.— We 

may  now  turn  to  consider  the  species  of  bacteria  found  in  the  soil. 
They  may  be  classified  in  five  main  groups ;  the  division  is  somewhat 
artificial,  but  convenient : — 

1.  The  Denitrifying  Bacteria. — This  group,  whose  function  has 
been  elucidated  largely  by  the  investigations  of  Professor  Warington, 
is   held  responsible  for  the  breaking   down  of   nitrates.     With  its 
members    may    be    associated    the    Decomposition    or    Putrefactive 
Bacteria,  which   break  clown  complex  organic  products  other  than 
nitrates  into  simpler  bodies. 

2.  The  Organisms  of  Nitrification. — To  this  group  belong  the  two 
chief  types  of  nitrifying  bacteria,  viz.,  those  which  oxidise  ammonia 
into  nitrites,  and  those  which  change  nitrites  into  nitrates. 

3.  The  Nitrogen-fixing  Bacteria,  found  mainly  in  the  nodules  on 
the  rootlets  of  certain  plants. 

4.  The  Common  Saprophytic  Bacteria,  whose  function  is  at  present 
but  imperfectly  known.     Many  are  putrefactive  germs. 

5.  The  Pathogenic  Bacteria. — This  division  includes  three  types, 
the  bacilli  of  tetanus,  malignant  oedema,  and  quarter  evil.    Under  this 
heading  we  shall  also  have  to  consider  in  some  detail  the  intimate 
relation  between  the  soil  and  such  important  bacterial  diseases  as 
tubercle  and  typhoid. 

To  enable  us  to  appreciate  the  work  which  the  "economic 
bacteria  "  perform,  it  will  be  necessary  to  consider  shortly  the  place 
they  occupy  in  the  economy  of  nature.  This  may  be  perhaps  most 
readily  accomplished  by  studying  the  scheme  shown  on  p.  120. 

The  threefold  function  of  ordinary  plant  life  is  nutrition,  assimi- 
lation, and  reproduction,  i.e.,  the  food  of  plants,  the  digestive  and 
storage  power  of  plants,  and  the  various  means  they  adopt  for  multi- 
plying and  increasing  their  species.  With  the  two  latter  we  have 
little  concern  in  this  place.  Eespecting  the  nutrition  of  plant  life, 
it  is  obvious  that,  like  animals,  plants  must  feed  and  breathe  to 
maintain  life.  Plant  food  is  of  three  chief  kinds,  viz.,  water,  inorganic 
salts,  and  gases.  Water  is  an  actual  necessity  to  the  plant  as  a  direct 
food  and  as  a  food-solvent,  i.e.  as  the  vehicle  of  important  inorganic 
materials.  The  hydrogen,  too,  of  the  organic  compounds  is  obtained 


120 


BACTERIA  IN  THE  SOIL 


from  the  decomposition  of  the  water  which  permeates  every  part  of 
the  plant,  and  is  derived  by  it  from  the  soil  and  from  the  aqueous 
vapour  in  the  atmosphere.  The  chief  inorganic  salts  of  which  proto- 
plasm is  constituted  are  composed  in  part  of  potassium,  magnesium, 
calcium,  iron,  phosphorus,  or  sulphur.  These  inorganic  elements  do 
not  enter  the  plant  as  such,  but  combined  with  other  substances  or 
dissolved  in  water.  Potassium,  calcium,  and  magnesium  are  absorbed 
chiefly  as  nitrates,  phosphates,  and  carbonates.  Iron  contributes  to 
the  formation  of  the  green  colouring-matter  of  plants,  indeed,  is 
essential  to  it,  and  is  also  derived  from  the  soil.  Phosphorus,  one  of 
the  chief  constituents  of  seeds,  generally  occurs  as  nucleo-albumin. 


A  SCHEME  SHOWING  THE  PLACE  AND  FUNCTION  OF  THE 
ECONOMIC  MICRO-ORGANISMS  FOUND  IN  SOIL 


Water. 

v   , 

Inorganic  Salts.                    Gases 
[Nitrates,  etc.].                     [CO.,,H,N 

1              / 

PLANT  LIFE. 

1 

,0]. 

Carbohydrates            Fats, 
[albumoses,  sugar, 
starch,  etc.]. 

Proteids             Vegetable 
[bodies  containing       Acids. 
Nitrogen]. 

Y 

ANIMAL  LIFE. 

I 

Mineral     Water. 
Salts. 

Gases  [CO.2,  etc.].            Water. 

Urea,  Albuminoids, 
Ammonia  compounds, 
etc. 

Nitrogen  in  many 
forms  locked  up 
in  the  body. 

DECOMPOSITION  AND  DENITRIFYING  BACTERIA. 
I 


Free  Nitrogen.         Gases  [CO2].         Water.  Ammonia,          [Nitrites]. 

and  other  elements 
of  broken-down 
complex  bodies. 

NITRIFYING   BACTERIA. 

Nitrites  [  =  Nitrous  organism 
|  (Nitrosomonas)]. 

Nitrates  [  —  Nitric  organism 
(Nitromonas,  or 
Nitrobacter)]. 
[In    soil    and 
available  for 
plant  life.  ] 


NlTROGEN-FlXING 

BACTERIA. 


[In  soil  and  in  the  nodules 
on  the  rootlets  of  Legu- 
minosce.  ] 


CONDITIONS  OF  PLANT  LIFE  121 

Sufyhur,  which  is  an  important  constituent  of  albumen,  is  derived 
from  the  sulphates  of  the  soil.  In  addition  to  the  above,  there  are 
other  elements,  sometimes  described  as  non-essential  constituents  of 
plants.  Amongst  these  are  silicon  (to  give  stiffness),  sodium,  chlorine, 
iodine,  bromine,  etc.  All  these  elements  contribute  to  the  formation 
or  quality  of  the  protoplasm  of  plants. 

The  gases  essential  to  plants,  and  absorbed  as  such,  are  two : 
Carbon  dioxide  (carbonic  acid)  and  Oxygen ;  the  necessary  hydrogen 
and  nitrogen  being  absorbed  in  the  form  of  salts.  By  the  aid  of  the 
green  chlorophyll  corpuscles,  and  under  the  influence  of  sunlight,  we 
know  that  leaves  absorb  the  carbon  dioxide  of  the  atmosphere,  and 
effect  certain  changes  in  it.  The  hydrogen,  as  we  have  seen,  is 
obtained  from  the  water.  Oxygen  is  absorbed  through  the  leaves  and 
through  the  root  from  the  interstices  of  the  soil.  Each  of  these  gases 
contributes  vitally  to  the  existence  of  the  plant.  The  fourth  gas,  nitro- 
gen, which  constitutes  more  than  two-thirds  of  the  air  we  breathe  (79 
per  cent,  of  the  total  volume  and  77  per  cent,  of  the  total  weight  of  the 
atmosphere),  is  also  an  absolutely  necessary  food  required  by  plants. 
Yet,  although  this  is  so,  the  plant  cannot  absorb  or  obtain  its  nitrogen 
in  the  same  manner  in  which  it  acquires  its  carbon — viz.,  by  absorption 
through  the  leaves — nor  can  the  plant  take  nitrogen  into  its  own 
substance  by  any  means  as  nitrogen.  Hence,  although  this  gas  is 
present  in  the  atmosphere  surrounding  the  plant,  the  plant  will 
perish  if  nitrogen  does  not  exist  in  some  combined  form  in  the  soil. 
Nitrates  and  compounds  of  ammonia  are  widely  distributed  in  nature, 
and  it  is  from  those  bodies  that  the  plant  obtains,  by  means  of  its 
roots,  the  necessary  nitrogen. 

Until  comparatively  recently  it  was  held  that  plant  life  could  not 
be  maintained  in  a  soil  devoid  of  nitrogen  or  compounds  thereof. 
But  it  has  been  found  that  certain  classes  of  plants  (the  Leguminosce 
for  example),  when  they  are  grown  in  a  soil  which  is  practically  free 
from  nitrogen  at  the  commencement,  do  take  up  this  gas  into  their 
tissues.  One  explanation  of  this  fact  is  that  free  nitrogen  becomes 
converted  into  nitrogen  compounds  in  the  soil  through  the  influence 
of  micro-organisms  present  there.  Another  explanation  attributes 
this  fixation  of  free  nitrogen  to  micro-organisms  existing  in  the 
rootlets  of  the  plant.  These  two  classes  of  organisms,  known  as  the 
nitrogen-fixing  organisms,  will  require  our  consideration  at  a  later 
stage.  Here  we  merely  desire  to  make  it  clear  that  the  main  supply 
of  this  gas,  absolutely  necessary  to  the  existence  of  vegetable  life 
upon  the  earth,  is  drawn  not  from  the  nitrogen  of  the  atmosphere, 
but  from  that  contained  in  nitrogen  compounds  in  the  soil.  The 
most  important  of  these  are  the  nitrates.  Here  then  we  have  the 
necessary  food  of  plants  expressed  in  a  sentence :  water,  inorganic  salts, 
gases  ;  some  of  the  salts  containing  nitrogen  in  the  form  of  nitrates. 


122  BACTERIA  IN  THE  SOIL 

Plant  life  seizes  upon  its  required  constituents,  and  by  means  of 
the  energy  furnished  by  the  sun's  rays  builds  these  materials  up  into 
its  own  complex  forms.  Its  many  and  varied  forms  fulfil  a  place  in 
beautifying  the  world.  But  their  contribution  to  the  economy  of 
nature  is,  by  means  of  their  products,  to  supply  food  for  animal  life. 
These  products  of  plant  life  are  chiefly  sugar,  starch,  fat,  and  proteids. 
Animal  life  is  not  capable  of  extracting  its  nutriment  from  soil,  but 
it  must  take  the  more  complex  foods  which  have  already  been  built 
up  by  vegetable  life.  Again,  the  complementary  functions  of  animal 
and  vegetable  life  are  seen  in  the  absorption  by  plants  of  one  of  the 
waste  materials  of  animals,  viz.,  carbonic  acid  gas.  Plants  abstract 
from  this  gas  carbon  for  their  own  use,  and  return  the  oxygen  to  the 
air,  which  in  its  turn  is  of  service  to  animal  life. 

By  animal  activity  some  of  these  foods  supplied  by  the  vegetable 
kingdom  are  at  once  decomposed  into  carbonic  acid  gas  and  water, 
which  goes  back  to  nature.  Much,  however,  is  built  up  still  further 
into  higher  and  higher  compounds.  The  proteids  are  converted  by 
digestion  into  more  soluble  forms,  such  as  albumoses  and  peptones ; 
these  in  their  turn  are  reconverted  into  less  soluble  proteids,  and 
become  assimilated  as  part  of  the  living  organism.  In  time  they 
become  further  changed  into  carbonic  acid,  sulphuric  acid,  water,  and 
certain  not  fully  oxidised  products,*  which  contain  the  nitrogen  of 
the  original  proteid.  In  the  table  these  bodies  have  been  represented 
by  one  of  their  chief  members,  viz.,  urea. 

It  is  clear  that  there  is  in  all  animal  life  a  double  process 
continually  going  on ;  there  is  a  building  up  (anabolism,  assimilation), 
and  there  is  a  breaking  down  (katabolism).  These  processes  will  not 
balance  each  other  throughout  the  whole  period  of  animal  life.  We 
have,  as  possibilities,  elaboration,  balance,  degeneration;  and  the 
products  of  animal  life  will  differ  in  degree  and  in  substance  accord- 
ing to  which  period  is  in  the  predominance.  These  products  we 
may  subdivide  simply  into  excretions  during  life  and  final  materials 
of  dissolution  after  death,  both  of  which  may  be  used  more  or  less 
immediately  by  other  forms  of  animal  or  vegetable  life,  or  immediately 
after  having  passed  to  the  soil.  We  may  shortly  summarise  the 
final  products  of  animal  life  as  carbonic  acid,  water,  and  nitrogenous 
remnants.  These  latter  will  occur  as  urea,  new  albumens,  compounds 
of  ammonia,  and  nitrogen  compounds  of  great  complexity  stored  up 
in  the  tissues  and  body  of  the  animal.  The  carbonic  acid,  water,  and 
other  simple  substances  like  them  will  return  to  nature  and  be  of 
immediate  use  to  vegetable  life.  But  otherwise  the  cycle  cannot  be 
completed,  for  the  more  complex  bodies  are  of  no  service  as  such 
to  plants  or  animals. 

*  E.  A.  Sehafer,  Text-hook  of  Physiology,  vol.  i.,  p.  25  (W.  D.  Halliburton). 


CONCERNED  WITH  PUTREFACTION  123 


1.  Decomposition  and  Denitrifieation 

In  order  that  this  complex  material  should  be  of  service  in  the 
economy  of  nature,  and  its  constituents  not  lost,  it  is  necessary  that 
it  should  be  broken  down  again  into  simpler  conditions.  This 
prodigious  task  is  accomplished  by  the  agency  of  two  groups  of 
organisms,  the  decomposition  and  denitrifying  *  bacteria.  The  organ- 
isms associated  with  decomposition  processes  are  numerous ;  some 
denitrify  as  well  as  break  down  organic  compounds.  This  group 
will  be  referred  to  under  "  Saprophytic  Bacteria."  The  reduction  by 
the  denitrifying  bacteria  may  be  simply  from  nitrate  to  nitrite,  or 
from  nitrate  to  nitric  or  nitrous  oxide  gas,  or  indeed  to  nitrogen 
itself.  In  all  these  processes  of  reduction  the  rule  is  that  a  loss  of 
nitrogen  is  involved.  How  that  free  nitrogen  is  brought  back  again 
and  made  subservient  to  plants  and  animals  we  shall  understand  at 
a  later  stage. 

Professor  Warington  has  set  forth  the  chief  facts  known  of 
this  decomposition  process.f  That  the  action  in  question  only 
occurs  in  the  presence  of  living  organisms  was  first  established 
by  Mensel  in  1875  in  natural  waters,  and  by  Macquenne  in  1882 
in  soils.  If  all  living  organisms  are  destroyed  by  sterilisation 
of  the  soil,  denitrification  cannot  take  place,  nor  can  vegetable 
life  exist.  "Bacteria  reduce  nitrates,"  says  Professor  Warington, 
"  by  bringing  about  the  combustion  of  organic  matter  by  the  oxygen 
of  the  nitrate,  the  temperature  distinctly  rising  during  the  operation." 
The  reduction  to  a  nitrite  is  a  common  property  of  bacteria.  But 
only  a  few  species  have  the  power  of  reducing  a  nitrate  to  gas. 
These  few  species  are,  however,  widely  distributed.  In  1886  Gay  on 
and  Dupetit  first  isolated  the  bacteria  capable  of  reducing  nitrates 
to  the  simplest  element,  nitrogen.  They  obtained  their  species  from 
sewage,  but  ten  years  later  denitrifying  bacteria  were  isolated  from 
manure.  That  soil  contains  a  number  of  these  reducing  organisms 
is  proved  by  introducing  a  particle  of  surface  soil  into  some  broth, 
to  which  has  been  added  1  per  cent,  of  nitre.  During  incubation  of 
such  a  tube  gas  is  produced,  and  the  nitrate  entirely  disappears. 

Whenever  decomposition  occurs  in  organic  substances  there  is  a 
reduction  of  compound  bodies,  and  in  such  cases  the  putrefying 
substances  obtain  their  decomposing  and  denitrifying  bacteria  from 
the  air.  The  chief  conditions  requisite  for  bringing  about  a  loss  of 
nitrogen  by  denitrification  are  enumerated  by  Professor  Warington 
as  follows: — (1)  the  specific  micro-organism;  (2)  the  presence  of  a 
nitrate  and  suitable  organic  matter;  (3)  such  a  condition  as  to 

*  "  Denitrifying"  means  reducing  nitrates. 

t  R.  Warington,  M.A.,  F.R.S.,  Jour.  Roy.  Agric.  Soc.  Eng.,  series  iii.,  vol.  viii. , 
part,  iv.,  p.  577  el  seq.     See  also  Trans.  Chem.  Soc.,  1884,  1888,  etc. 


124  BACTERIA  IN  THE  SOIL 

aeration  that  the  supply  of  atmospheric  oxygen  shall  not  be  in  excess 
relatively  to  the  supply  of  organic  matter;  (4)  the  usual  essential 
conditions  of  bacterial  growth.  "  Of  these,"  he  says,  "  the  supply  of 
organic  matter  is  by  far  the  most  important  in  determining  the 
extent  to  which  denitrification  will  take  place."  The  necessarily 
somewhat  unstable  condition  facilitates  its  being  split  up  by  means 
of  bacteria.  The  bacteria  in  their  turn  are  ready  to  seize  upon  any 
products  of  animal  life  which  will  serve  as  their  food.  Thus,  by 
reducing  complex  bodies  to  simple  ones,  these  denitrifying  organ- 
isms act  as  the  necessary  link  to  connect  again  the  excretions  of 
the  animal  body,  or  after  death  the  animal  body  itself,  with  the 
soil. 

In  a  book  of  this  nature  it  has  been  deemed  advisable  not  to 
enter  into  minute  description  of  all  the  species  of  bacteria  mentioned. 
Some  of  the  chief  are  described  more  or  less  fully.  We  cannot, 
however,  do  more  than  name  several  of  the  chief  organisms 
concerned  in  reducing  and  breaking  down  compounds.  As  we  shall 
find  in  the  bacteria  of  nitrification,  so  also  here,  the  entire  process 
is  rarely,  if  ever,  performed  by  one  species.  There  is  indeed  a 
remarkable  division  of  labour,  not  only  between  decomposition 
bacteria  and  denitrification  bacteria,  but  between  different  species 
of  the  same  group.  Bacillus  fluorescens  non-liquefaciens,  My  co- 
derma  urece,  and  some  of  the  staphylococci  break  down  nitrates 
(denitrification),  and  also  decompose  other  compound  bodies.  Amongst 
the  group  of  putrefactive  bacteria  found  in  soil  may  be  named  B. 
coli,  B.  mycoides,  B.  mesentericus,  B.  liquidus,  B.  prodigiosus,  B. 
ramosus,  B.  vermicularis,  B.  liquefaciens,  and  many  members  in  the 
great  family  of  Proteus.  Some  perform  their  function  in  soil,  others 
in  water,  and  others,  again,  in  dead  animal  bodies.  Dr  Buchanan 
Young,  to  whose  researches  in  soil  we  have  referred,  has  pointed  out 
that  in  the  upper  reaches  of  burial  soil,  where  these  bacteria  are 
most  largely  present,  there  is  as  a  result  no  excess  of  organic  carbon 
and  nitrogen.  Even  in  the  lower  layers  of  such  soil  it  is  rapidly 
broken  down. 

It  will  be  observed,  from  a  glance  at  the  table  (p.  120),  that  the 
chief  results  of  decomposition  and  denitrification  are  as  follow :  free 
nitrogen,  carbonic  acid  gas,  and  water,  ammonia  bodies,  and  some- 
times nitrites.  The  nitrogen  passes  into  the  atmosphere,  and  is 
"lost";  the  carbonic  acid  and  water  return  to  nature,  and  are  at 
once  used  by  vegetation.  The  ammonia  and  nitrites  await  further 
changes.  These  further  changes  become  necessary  on  account  of  the 
fact,  already  discussed,  that  plants  require  their  nitrogen  to  be  in  the 
form  of  nitrates  in  order  to  use  it.  Nitrates  obviously  contain  a 
considerable  amount  of  oxygen,  but  ammonia  contains  no  oxygen,  and 
nitrites  very  much  less  than  nitrates.  Hence  a  process  of  oxidation 


CONCERNED  WITH  NITRIFICATION  125 

is  required  to  change  the  ammonia  into  nitrites  and  the  nitrites  into 
nitrates. 

2.  Nitrification 

This  oxidation  is  performed  by  the  nitrifying  micro-organisms, 
and  the  process  is  known  as  nitrification.  It  should  be  clearly 
understood  that  the  process  of  nitrification  may,  so  to  speak,  dovetail 
with  the  process  of  denitrification.  No  exact  dividing  line  can  be 
drawn  between  the  two,  although  they  are  definite  and  different 
processes.  In  a  carcase,  for  example,  both  processes  may  be  going 
on  concomitantly,  so  also  in  manure.  There  is  no  hard-and-fast  line 
to  be  drawn  in  the  present  state  of  our  knowledge.  Other  organisms 
beside  the  true  nitrification  bacteria  may  be  playing  a  part,  and  it  is 
impossible  exactly  to  measure  the  action  of  the  latter,  where  they 
began  and  where  the  preliminary  attack  upon  the  nitrogenous  com- 
pounds terminated.  In  all  cases,  however,  according  to  Professor 
Warington,  the  formation  of  ammonia  has  been  found  to  precede  the 
formation  of  nitrous  or  nitric  acid. 

It  was  Pasteur  who  (in  1862)  first  suggested  that  the  production 
of  nitric  acid  in  soil  might  be  due  to  the  agency  of  germs,  and  it  is 
to  Schlosing  and  Miintz  that  the  credit  belongs  for  first  demonstrat- 
ing (in  1877)  that  the  true  nature  of  nitrification,  the  conversion  of 
ammonia  into  nitric  acid,  depended  upon  the  activity  of  a  living 
micro-organism.*  Partly  by  Schlosing  and  Miintz  and  partly  by 
Warington  (who  was  then  engaged  in  similar  work  at  Rothamsted),  it 
was  later  established  (1)  that  the  power  of  nitrification  could  be  com- 
municated to  substances  which  had  not  hitherto  nitrified  by  simply 
seeding  them  with  a  nitrified  substance,  and  (2)  that  the  process  of 
nitrification  in  garden  soil  was  entirely  suspended  by  the  vapour  of 
chloroform  or  carbon  disulphide.  The  conditions  for  nitrification, 
the  limit  of  temperature,  and  the  necessity  of  plant  food,  have 
furnished  additional  proof  that  the  process  is  due  to  a  living  organism. 
These  conditions,  according  to  Warington,  are  as  follows: — 

1.  Food  (of  which  phosphates  are  essential  constituents).     "The 
nitrifying  organism  can  apparently  feed  upon  organic  matter,  but  it 
can  also,  apparently  with  equal  ease,  develop  and  exercise  all  its 
functions  upon  purely  inorganic  food "  (J.  M.  H.  Munro).f     Wino- 
gradsky   prepared   vessels    and    solutions    carefully   purified   from 
organic  matter,  and  these  solutions  he  sowed  with  the  nitrifying 
organism,  and  found  that  they  flourished.     Professor  Warington  has 
employed  the  acid  carbonates  of  sodium  and  calcium  with  distinct 
success  as  ingredients  of  an  ammoniacal  solution  undergoing  nitrifi- 
cation. 

2.  The  next  condition  of  nitrification  is  the  presence  of  oxygen. 

*  Compt.  Rend.,  1877,  pp.  84,  301.  t  Trans.  Chem.  Soc.y  1886,  etc. 


126  BACTERIA  IN  THE  SOIL 

Without  it  the  reverse  process,  denitrification,  occurs,  and  instead 
of  a  building  up  we  get  a  breaking  down,  with  an  evolution  of 
nitrogen  gas.  The  amount  of  oxygen  present  has  an  intimate  pro- 
portion to  the  amount  of  nitrification,  and  with  16  to  21  per  cent,  of 
oxygen  present  the  nitrates  are  more  than  four  times  as  much  as 
when  the  smallest  quantity  of  oxygen  is  supplied.  The  use  of  tillage 
in  promoting  nitrification  is  doubtless  in  part  due  to  the  aeration  of 
the  soil  thus  obtained. 

3.  A  third  condition  is  the  presence  of  a  base  with  which  nitric 
acid  when  formed  may  combine.     Nitrification  can  only  take  place 
in  a  feebly  alkaline  medium,  but  an  excess  of  alkalinity  will  retard 
the  process. 

4.  The  last  requirement  is  a  favourable  temperature.     As  low  as 
37°  or  39°  F.  (3-4°  C.)  will  suffice,  but  at  a  higher  temperature  it 
becomes  much  more  active.     According  to  Schlosing  and  Miintz,  at 
54°  F.  (12°  C.)  nitrification  becomes  really  active,  and  it  increases 
as  the  temperature  rises  to  99°  F.  (37°  C.),  after  which  it  falls.     A 
high  temperature  or  a  strong  light  are  prejudicial  to  the  process. 

We  are  now  in  a  position  to  consider  shortly  some  of  the  char- 
acters of  these  nitrification  bacteria.  They  may  readily  be  divided 
into  two  chief  groups,  not  in  consideration  of  their  form  or  biological 
characteristics,  but  on  account  of  the  duties  which  they  perform. 
Just  as  we  observed  that  there  were  few  denitrifying  organisms 
which  could  break  down  ammonia  compounds  to  nitrogen  gas,  so  is  it 
also  true  that  there  are  few  nitrifying  bacteria  which  can  build  up 
from  ammonia  to  the  nitrates.  Nature  has  provided  that  this  shall 
be  accomplished  in  two  stages,  viz.,  a  first  stage  from  ammonia  bodies 
to  nitrites  (nitrosification),  and  a  second  stage  from  nitrites  to 
nitrates.  The  agent  of  the  former  is  termed  the  nitrous  organism, 
the  latter  the  nitric  organism.  Both  are  contributing  to  the  final 
production  of  nitrates  which  can  be  used  by  plant  life.* 

The  Nitrous  Organism  (Mtrosomonas).  Prior  to  Koch's  gelatine 
method  the  isolation  of  this  bacterium  proved  an  exceedingly  difficult 
task.  But  even  the  adoption  of  this  isolating  method  seemed  to  give 
no  better  results,  and  for  an  excellent  reason :  the  nitrifying 
organisms  will  not  grow  on  gelatine.  To  Winogradsky  f  and  Percy 
FranklandJ  belongs  the  credit  of  separately  isolating  the  nitrous 
organism  on  the  surface  of  gelatinous  silica  containing  the  necessary 

*  The  saltpetre  beds  of  Chili  and  Peru  are  an  excellent  example  of  the  applica- 
tion of  these  facts.  Nitrates  are  there  produced  from  the  faecal  evacuations  of  sea- 
fowl  in  such  quantities  as  to  form  an  article  of  commerce.  A  like  form  of  utilisation 
of  the  action  of  these  bacteria  was  once  practised  on  the  continent  of  Europe. 
Considerable  nitrate  deposits  have  recently  been  discovered  in  California,  Economic 
application  is  also  seen  in  the  treatment  of  sewage  referred  to  elsewhere. 

f  Ann.  de  VInst.  Pasteur,  1890,  p.  213. 

•$.Phil.  Trans.  Roy.  Soc.,  1890,  B.  107. 


THE  NITROUS  ORGANISM  127 

inorganic  food.  Professor  Warington,  in  his  lectures  under  the  Lawes 
Agricultural  Trust,  has  described  this  organism  as  follows : — 

"  The  organism  as  found  in  suspension  in  a  freshly  nitrified  solu- 
tion consists  largely  of  nearly  spherical  corpuscles,  varying  extremely 
in  size.  The  largest  of  these  corpuscles  barely  reaches  a  diameter  of 
one-thousandth  of  a  millimetre,  and  some  are  so  minute  as  to  be 
hardly  discernible  in  photographs.  The  larger  ones  are  frequently 
not  strictly  circular,  and  are  sometimes  seen  in  the  act  of  dividing. 

"Besides  the  form  just  described,  there  is  another,  not  universally 
present  in  solutions,  in  which  the  length  is  considerably  greater  than 
its  breadth.  The  shape  varies,  being  occasionally  a  regular  oval,  but 
sometimes  largest  at  one  end,  and  sometimes  with  the  ends  truncated. 
The  circular  organisms  are  probably  the  youngest. 

"  This  organism  grows  in  broth,  diluted  milk,  and  other  solutions 
without  producing  turbidity.  When  acting  on  ammonia  it  produces 
only  nitrites.  It  is  without  action  on  potassium  nitrite.  It  is,  in 
fact,  the  nitrous  organism  which,  as  we  have  previously  seen,  may  be 
separated  from  soil  by  successive  cultivations  in  ammonium  carbonate 
solution."* 

The  elongated  forms  appear  to  be  a  sign  of  arrested  growth. 
Normally,  the  organism  is  about  1'8  /x  long,  or  nearly  three  times  as 
long  as  the  nitric  organism.  It  possesses  a  gelatinous  capsule.  "  The 
motile  cells,  stained  by  Loffler's  method,  are  seen  to  have  a  flagellum 
in  the  form  of  a  spiral."  When  grown  on  silica  jelly  the  nitrous 
organism  appears  in  the  same  two  forms — zooglea  and  free  cells — as 
when  cultivated  in  a  fluid.  It  commences  to  show  growth  in  about 
four  days,  and  is  at  its  maximum  on  about  the  tenth  day.  Upon 
gypsum,  to  which  1  per  cent,  of  magnesium  carbonate  has  been  added, 
the  organism  grows  in  the  same  form  of  small  brown  colonies,  but 
more  rapidly.  Winogradsky  found  that  there  were  considerable 
differences  in  the  morphology  of  the  organism  according  to  the  soil 
from  which  it  was  taken.  The  solution  used  by  him  consisted  of 
water  containing  1  per  1000  ammonium  sulphate,  1  per  1000  potas- 
sium phosphate,  and  1  per  100  magnesium  carbonate. 

As  we  have  already  seen,  an  astonishing  property  of  this  organism 
is  its  ability  to  grow  and  perform  its  specific  function  in  solutions 
absolutely  devoid  of  organic  matter  (Munro).  Some  authorities  hold 
that  it  acquires  its  necessary  carbon  from  carbonic  acid.  The  mode 
of  ctilturing  it  was  as  follows : — To  sterilised  flat-bottomed  flasks  add 
100  c.c.  of  a  solution  made  of  two  grams  of  ammonium  sulphate,  one 
gram  of  potassium  phosphate,  and  1000  c.c.  of  distilled  water.  To 
this  was  added  half  a  gram  of  magnesium  sulphate,  two  grams  of 
common  salt,  and  04  of  a  gram  of  ferrous  sulphate.  Now  the  flask 

*  U.S.A.  Dept.  of  Agriculture:  Lectures  under  the  Lawes  Agricultural  Trust. 
By  Robert  Warington,  T.R.S.,  1891,  pp.  58,  59. 


128  BACTERIA  IN  THE  SOIL 

was  inoculated  with  a  small  portion  of  the  soil  under  investigation, 
and  after  four  or  five  days  sub -cultured  on  the  same  medium  in  fresh 
flasks,  and  repeated  half  a  dozen  times.  Now,  as  this  inorganic 
medium  was  unfavourable  to  the  ordinary  bacteria  of  soil,  it  was 
supposed  that  after  several  sub-cultures  the  nitrous  organism  was 
isolated  in  pure  culture.  Winogradsky  employed  for  sub-culturing 
upon  a  solid  medium  a  mineral  gelatine,  silica  jelly.*  Upon  this 
medium  it  is  possible  to  sub-culture  a  pure  growth  from  the  film  at 
the  bottom  of  the  flasks  in  which  the  nitrous  organism  is  first 
isolated.  In  1899  Winogradsky  showed  that  the  nitrous  organism 
(nitroso-bacterium)  was  able  to  grow  in  the  presence  of  large  amounts 
of  organic  matter,  and  since  that  date  Fremlin  has  carried  this  branch 
of  work  to  a  further  stage  of  advancement.  He  has  shown  that 
cultures  developed  in  inorganic  solutions  become  eventually  pure 
cultures  of  this  species  of  nitrifying  organism,  and  when  inoculated 
into  solutions  containing  small  quantities  of  organic  matter  they  were 
able  to  oxidise  the  ammonia  present.  Fremlin  has  also  demonstrated 
that  the  nitrous  organism  grows  well  on  silica  jelly  and  ammonia  agar, 
and  colonies  from  these  media  transferred  to  beef-broth  agar  and 
gelatine  also  grew  well.  From  these  experiments  he  concluded  "  that 
the  nitroso-bacterium  grows  well  on  any  ordinary  medium  "  but  "  that 
in  the  presence  of  large  percentages  of  organic  matter  the  nitroso- 
bacterium,  although  growing  very  profusely,  loses  for  a  time  the  power 
of  converting  ammonia  into  nitrites."f 

The  Nitric  Organism. — It  was  soon  learned  that  the  nitrous 
organism,  even  when  obtainable  in  large  quantities  and  in  pure 
culture,  was  not  able  entirely  to  complete  the  nitrifying  process.  As 
early  as  1881  Professor  Warington  had  observed  that  some  of  his 
cultures,  though  capable  of  changing  nitrites  into  nitrates,  had  no 
power  of  oxidising  ammonia. .  These  he  had  obtained  from  advanced 
sub-cultures  of  the  nitrous  organism,  and  somewhat  later  Wino- 
gradsky isolated  and  described  this  companion  of  the  nitrous 
organism.  It  develops  freely  in  solutions  to  which  no  organic  matter 
has  been  added ;  indeed,  much  organic  matter  will  prevent  it  growing.J 

The   temperature   for  incubation  is  30°   C.     Winogradsky  con- 

*  Two  per  cent,  of  dialysed  silicic  acid  mixed  with  neutral  salts  and  magnesium 
carbonate  in  order  to  solidify  it. 

t  Jour,  of  Hyg.,  1903,  pp.  378,  379. 

J  Compt.  Rend.,  113  (1891)  p.  89 — Winogradsky  isolated  it  from  soils  from  various 
parts  of  the  world  on  the  following  medium  :— Water,  lOOO'O  ;  potassium  phosphate, 
1*0;  magnesium  sulphate,  0*5;  calcium  chloride,  a  trace;  sodium  chloride,  2'0. 
About  20  c.c.  of  this  solution  was  placed  in  a  flat-bottom  flask,  and  a  little  freshly 
washed  magnesium  carbonate  was  added.  The  flask  was  closed  with  cotton-wool, 
and  the  whole  sterilised.  To  each  flask  2  c.c.  of  a  2  per  cent,  solution  of  ammonium 
sulphate  was  subsequently  added.  Recently,  the  following  medium  has  been  used 
for  cultivation  of  the  nitric  organism  : — Sodium  nitrite,  I'O  ;  sodium  carbonate,  I'D ; 
sodium  chloride,  0'5;  potassium  phosphate,  O'o  ;  magnesium  sulphate,  0'3;  ferrous 
sulphate,  0*4,  in  1000  parts  of  distilled  water. 


PLATE  11. 


NITROUS  ORGANISM. 
x  1000. 


NITRIC  ORGANISM. 
x   1000. 


NlTROGKN-FlXING    ORGANISMS    FROM    SECRETIONS    OF    ROOT-NODULES   TAKEN   FROM    LEGUMINOS^. 

Film  preparations,     x  1000. 


[To  face\)pagc  128. 


THE  NITRIC  ORGANISM  129 

eluded  that  the  oxidation  of  nitrites  to  nitrates  was  brought  about  by 
a  specific  organism  independently  of  the  nitrous  organism.  He  suc- 
cessfully sub-cultured  it  from  his  inorganic  medium  on  to  silica  jelly 
and  also  on  to  purified  agar.  He  believes  the  organism,  like  its  com- 
panion, derives  its  nutriment  solely  from  inorganic  matter,  but  this 
is  not  finally  established. 

The  form  of  the  nitric  organism  (or  nitromonas,  as  it  was  once 
termed)  is  allied  to  the  nitrous  organism.  The  cells  are  elongated, 
rarely  oval,  but  sometimes  pear-shaped.  They  are  more  than  half  a 
micromillimetre  in  length,  and  somewhat  less  in  thickness.  The 
cells  have  a  gelatinous  membrane.  Like  the  other  nitrifying  bacteria, 
its  development  and  action  are  favoured  by  the  presence  of  the  acid 
carbonates  of  calcium  and  sodium.  Of  the  latter,  six  grams  per  litre 
or  even  a  smaller  quantity  gives  good  results.  The  sulphate  of 
calcium  can  be  used,  but  the  organism  prefers  the  carbonates.  The 
differences  between  these  two  bacteria  are  small,  with  the  exception 
of  their  chemical  action.  The  nitric  organism  has  no  action  upon 
ammonia,  and  its  presence  in  very  small  amount  (five  parts  per 
million)  hinders  its  development,  and  in  sixty-four  parts  per  million 
prevents  its  action  on  a  nitrite.* 

We  may  here  summarise  the  general  facts  respecting  nitrification. 
Winogradsky  proposes  to  term  the  group  nitroso-lacteria,  and  to 
classify  thus: — 

'Nitrosomonas,  containing  at  least  two 


Nitrous  organisms    =    - 


species,  viz.,  the  European  and  the 
Java. 


Nitrosococcus. 
Nitric  organism        =       Nitrobacter. 

Nitrification  occurs  in  two  stages,  each  stage  performed  by  a 
distinct  organism.  By  one  (nitrosomonas)  ammonia  is  converted  into 
nitrite;  by  the  other  (nitrobacter)  the  nitrite  is  converted  into 
nitrate.f  Both  organisms  are  widely  and  abundantly  distributed  in 

*  The  course  of  nitrification  maybe  followed  by  means  of  chemical  tests.  1. 
The  disappearance  of  ammonia.  2.  The  appearance  of  nitrite.  3.  Its  disappear- 
ance. 4.  Appearance  of  nitrate. 

t  Professor  Warington,  in  Report  IV.  (p.  526)  of  his  admirable  series  of  papers 
on  the  subject,  draws  attention  to  Miintz's  criticism  that  the  nitrifying  organisms 
only  oxidise  from  nitrogenous  matter  to  nitrites,  and  not  from  nitrites  to  nitrates. 
Miintz  held  that  the  conversion  of  nitrite  into  nitrate  is  brought  about  by  the  joint 
action  of  carbonic  acid  and  oxygen.  Professor  Warington's  experiments,  however, 
clearly  illustrate  that  the  production  of  nitrates  from  nitrites  in  an  ammoniacal  solu- 
tion can  be  determined  by  the  character  of  the  bacterial  culture  with  which  the 
solution  is  seeded,  and  that  in  a  solution  of  potassium  nitrite  conversion  into 
nitrate  can  be  determined  by  the  introduction  of  the  nitric  organism.  Professor 
Warington  still  adheres  to  the  opinion,  in  favour  of  which  he  has  produced  so  much 
evidence,  that  the  formation  of  nitrates  in  the  soil  is  due  to  the  nitric  organism 
which  soil  always  contains. 


130  BACTERIA  IN  THE  SOIL 

the  superficial  soils.  They  act  together  and  in  conjunction,  and  for 
one  common  purpose.  They  are  separable  by  employing  favourable 
media.  "  If  we  employ  a  suitable  inorganic  solution  containing  potas- 
sium nitrite,  but  no  ammonia,  we  shall  presently  obtain  the  nitric 
organism  alone,  the  nitrous  organism  feeding  on  ammonia  being 
excluded.  If,  on  the  other  hand,  we  employ  an  ammonium  carbonate 
solution  of  sufficient  strength,  we  have  selected  conditions  very  un- 
favourable to  the  growth  of  the  nitric  organism,  and  a  few  cultiva- 
tions leave  the  nitrous  organism  alone  in  possession  of  the  field "  * 
(Warington).  Adeney  has  summarised  conclusions  respecting 
nitrification  as  follows:  (1)  In  organic  solutions  containing  ammonia 
nitrous  organisms  thrive,  but  nitric  organisms  gradually  lose  their 
vitality ;  (2)  nitrous  organisms  cannot  oxidise  nitrites  to  nitrates  in 
inorganic  solutions ;  (3)  nitric  organisms  thrive  in  inorganic  solutions 
containing  nitrites;  (4)  the  presence  of  peaty  or  humous  matter 
appears  to  preserve  the  vitality  of  nitric  organisms  during  the 
fermentation  of  ammonia,  and  establishes  conditions  whereby  it  is 
possible  for  the  nitric  organisms  to  thrive  simultaneously  in  the 
same  solution  as  the  nitrous  organisms.  Other  conditions  necessary 
for  nitrification  are,  of  course,  the  presence  of  ammonia  preceding 
the  appearance  of  nitrous  or  nitric  acid,  the  presence  of  a  fixed  base, 
not  too  high  a  degree  of  alkalinity,  and  darkness  and  free  admission 
of  air. 

A  word  may  be  said  upon  the  natural  distribution  of  these  nitrify- 
ing bacteria  before  we  leave  them.  They  belong  to  the  soil,  river- 
water,  and  sewage.  They  are  also  said  to  be  frequently  present  in 
well-water.  From  experiments  at  Eothamsted  it  appears  that  the 
organisms  occur  mostly  in  the  first  12  inches,  in  subsoils  of  clay  down 
to  3  or  4  feet,  and  in  sandy  soils  probably  at  even  a  greater  depth. 
These  facts  are  of  the  first  importance  in  relation  to  the  biological 
treatment  of  sewage. 

We  have  now  given  some  consideration  to  the  chief  events  in  the 
life-cycle  of  nature  depicted  in  the  table  (p.  120).  There  is  but  one 
further  process  in  which  bacteria  play  a  part,  and  which  requires  some 
mention.  It  will  have  been  noticed  that  at  certain  stages  in  the 
cycle  there  is  a  more  or  less  appreciable  "  loss  "  of  free  nitrogen.  In 
the  process  of  decomposition  brought  about  by  the  denitrifying 
bacteria,  a  very  considerable  portion  of  the  nitrogen  is  dissipated 
into  the  air  in  the  form  of  a  free  gas.  This  is  the  last  stage  of  all 
proteid  decomposition,  so  that  wherever  putrefaction  is  going  on 
there  is  a  continual  "loss"  of  an  element  essential  to  life.  Thus  it 
would  appear  at  first  sight  that  the  sum-total  of  nitrogen  food  must 
be  diminishing. 

But  there  are  other  ways  also  in  which  nitrogen  is  being  set  free. 
*  Waives  Agricultural  Trust  Lectures,  1891,  p.  63. 


NITROGEN-FIXING  BACTERIA  131 

In  the  ordinary  processes  of  vegetation  there  is  a  gradual  draining  of 
the  soil  and  a  passing  of  nitrogen  into  the  sea;  the  products  of 
decomposition  pass  from  the  soil  by  this  drainage,  and  are  "  lost "  as 
far  as  the  soil  is  concerned.  Many  of  the  methods  of  sewage  dis- 
posal are  in  reality  depriving  the  land  of  the  return  of  nitrogen, 
which  is  its  necessity.  Again,  nitrogen  is  freed  in  explosions  of 
gunpowder,  nitroglycerine,  and  dynamite,  for  whatever  purpose  they 
are  used.  Hence  the  great  putrefactive  "  loss  "  of  nitrogen,  with  its 
subsidiary  losses,  contributes  to  reduce  this  essential  element  of  all 
life,  and  if  there  were  no  method  of  bringing  it  back  again  to  the 
soil,  it  would  seem  that  plant  life,  and  therefore  animal  life,  would 
speedily  terminate. 

3.  Nitrogen-Fixing*  Bacteria 

It  is  at  this  juncture,  and  to  perform  this  vital  function, 
that  the  nitrogen-fixing  bacteria  play  their  wonderful  part:  they 
help  to  recover  the  free  nitrogen  and  fix  it  in  the  soil.  Excepting  a 
small  quantity  of  combined  nitrogen  coming  down  in  rain  and  in 
minor  aqueous  deposits  from  the  atmosphere,  the  great  source  of  the 
nitrogen  of  vegetation  is  the  store  in  the  soil  and  subsoil,  whether 
derived  from  previous  accumulations  or  from  recent  supplies  by 
manure. 

Sir  William  Crookes  has  pointed  out  the  vast  importance  of 
using  all  the  available  nitrogen  in  the  service  of  wheat  production.* 
The  distillation  of  coal  in  the  process  of  gas-making  yields  a  certain 
amount  of  its  nitrogen  in  the  form  of  sulphate  of  ammonia,  and  this, 
like  other  nitrogenous  manures,  might  be  used  to  give  back  to  the 
soil  some  of  the  nitrogen  drained  from  it.  But  such  manuring 
cannot  keep  pace,  according  to  Sir  W.  Crookes,  with  the  present 
loss  of  fixed  nitrogen  from  the  soil.  We  have  already  referred  to 
several  ways  in  which  "  loss  "  of  nitrogen  occurs.  To  these  may  well 
be  added  the  enormous  loss  occurring  in  the  waste  of  sewage  when  it 
is  passed  into  the  sea.  As  the  President  of  the  British  Association 
pointed  out,  the  more  widely  this  wasteful  system  is  extended, 
recklessly  returning  to  the  sea  what  we  have  taken  from  the  land, 
the  more  surely  and  quickly  will  the  finite  stocks  of  nitrogen,  locked 
up  in  the  soils  of  the  world,  become  exhausted.  Let  us  remember 
that  the  plant  creates  nothing  in  this  direction  ;  there  is  no  com- 
bined nitrogen  in  wheat  which  is  not  absorbed  from  the  soil,  and 
unless  the  abstracted  nitrogen  is  returned  to  the  soil,  its  fertility 
must  be  ultimately  exhausted.  When  we  apply  to  the  land  sodium 
nitrate,  sulphate  of  ammonia,  guano,  and  similar  manurial  substances, 
we  are  drawing  on  the  earth's  capital,  and  our  drafts  will  not  be 

*  The  Wheat  Problem,  1899. 


132  BACTERIA  IN  THE  SOIL 

perpetually  responded  to.*  We  know  that  a  virgin  soil  cropped  for 
several  years  loses  its  productive  powers,  and  without  artificial  aid 
becomes  unfertile.  For  example,  through  this  exhaustion  forty 
bushels  of  wheat  per  acre  have  dwindled  to  seven.  Eotation  of  crops 
is  an  attempt  to  meet  the  problem,  and  the  four-course  rotation  of 
turnips,  barley,  clover,  and  wheat  witnesses  to  the  fact  that  practice 
has  been  ahead  of  science  in  this  matter.  It  is  unnecessary  to  add 
that  rotation  of  crops  and  the  use  of  the  Leguminosse  does  not  absolve 
the  agriculturist  from  maintaining  the  land  in  ripe  condition  by 
manuring  and  ordinary  tillage. 

The  store  of  nitrogen  in  the  atmosphere  is  practically  unlimited, 
but  it  is  fixed  and  rendered  assimilable  only  by  organic  processes  of 
extreme  slowness.  We  may  shortly  glance  at  these,  for  it  is  upon 
these  processes,  plus  a  return  to  the  soil  of  sewage,  that  we  must 
depend  in  the  future  for  storing  nitrogen  as  nitrates. 

1.  Some  combined  nitrogen  is  absorbed  by  the  soil  or  plant  from 
the  air,  for  example,  fungi,  lichens,  and  some  algae,  and  the  absorption 
is  in  the  form  of  ammonia  and  nitric  acid.     This  is  admittedly  a 
small  quantity. 

2.  Some  free  nitrogen  is  fixed  within  the  soil  by  the  agency  of 
porous  and  alkaline  bodies. 

3.  Some,  again,  may  be  assimilated  by  the  higher  chlorophyllous 
plants  themselves,  independently  of  bacteria  (Frank). 

4.  Electricity  fixes,  and  may  in  the  future  be  made  to  fix  more, 
nitrogen.     If  a  strong  inducive  current  be  passed  between  terminals, 
the  nitrogen  from  the  air  enters  into  combination  with  the  oxygen, 
producing  nitrous  and  nitric  acids. 

5.  Abundant  evidence  has  now  been  produced  in  support  of  the 
fact  that  there  is  considerable  fixation  by  means  of  bacteria. 

Bacterial  life  in  several  ways  is  able  to  reclaim  from  the  atmo- 
sphere this  free  nitrogen,  which  would  otherwise  be  lost.  The  first 
method  to  which  reference  may  be  made  is  that  involving 
symbiosis.  This  term  signifies  "  a  living  together "  of  two  different 
forms  of  life,  generally  for  a  specific  purpose.  Marshall  Ward  has 
recently  defined  it  as  the  co-operation  of  two  associated  organisms  to 
their  mutual  advantage,  each  symbiont  being  incapable  of  carrying 
on  alone  the  work  which  the  symbiotic  association  is  able  to  per- 
form.f  It  is  convenient  to  restrict  the  term  symbiosis  to  comple- 
mentary partnerships  such  as  exist  between  algoid  and  fungoid 
elements  in  lichens,  or  between  unicellular  algae  and  Badiolarians,J 

*  Sir  John  Lawes  and  Sir  Henry  Gilbert  (Times,  2nd  December  1898)  have 
pointed  out  that  the  addition  of  nitrates  only  would  be  of  no  permanent  use  to  the 
wheat  crop.  They  rely  upon  thorough  tillage  and  proper  rotation  of  crops  as  the 
means  of  improving  the  nitrogen  value  of  the  soil. 

f  British  Association  for  the  Advancement  of  Science  Report,  1899,  p.  693. 

J  Geddes,  Nature,  xxv.,  1882. 


SYMBIOSIS 


133 


or  between  bacteria  and  higher  plants.  The  partnerships  between 
hermit  crabs  and  sea-anemones  and  the  like  are  sometimes  defined 
by  the  term  commensalism  (joint  diet),  which  is  applied  to  such 
associations  having  negative  results,  neither  partner  gaining  much 
advantage  from  the  association.  Symbiosis  and  commensalism  must 
be  distinguished  from  parasitism,  which  indicates  that  all  the 
advantage  is  on  the  side  of  the  parasite,  and  nothing  but  loss  on 
the  side  of  the  host.  Association 
of  organisms  together  for  increase 
of  virulence  and  function  should 
be  distinguished  from  symbiosis, 
and  mere  existence  of  two  or  more 
species  of  bacteria  in  one  medium 
is  not,  of  course,  symbiosis.  Most 
frequently  such  a  condition  would 
result  in  injury  and  the  subsequent 
death  of  the  weaker  partner,  an 
effect  precisely  opposite  to  that  de- 
lined  by  this  term. 

The  example  of  bacteriological 
symbiosis  with  which  we  are  con- 
cerned here  is  that  partnership  be- 
tween bacteria  and  some  of  the 
higher  plants  (Leguminosse)  for  the 
purpose  of  fixing  nitrogen  in  the 
plant  and  in  the  surrounding  soil.* 

The  nitrogen-fixing-  bacteria, 
the  third  group  of  micro-organisms 
connected  with  the  soil,  exist  in 
groups  and  colonies  situated  inside 
the  nodules,  appearing,  under  cer- 
tain circumstances,  on  the  rootlets 
of  the  pea,  bean,  and  other  Legu- 
ininos?e.  It  was  Hellriegel  and 
Wilfarth  who  first  pointed  out 
that,  although  the  higher  chloro- 
phyllous  plants  could  not  directly 
obtain  or  utilise  free  nitrogen, 
some  of  them  at  any  rate  could  acquire  nitrogen  brought  into  com- 
bination under  the  influence  of  bacteria.  Hellriegel  found  that  the 
gramineous,  polygonaceous,  cruciferous,  and  other  orders  depended 

*  Examples  of  bacteria  symbionts  are  numerous  ;  e.g.  the  dissolution  of  cellulose 
(Van  Senus) ;  the  decomposition  of  sound  potato  in  water  exhausted  of  air  (Ward) ; 
the  reduction  of  sulphates  ;  the  oxidation  of  sulphuretted  hydrogen  ;  the  iron 
bacteria,  etc. 


FIG.  19.— Rootlet  of  Pea  with  Nodules. 


134  BACTERIA  IN  THE  SOIL 

upon  combined  nitrogen  supplied  within  the  soil,  but  that  the 
Leguminosge  did  not  depend  entirely  upon  such  supplies. 

It  was  observed  that  in  a  series  of  pots  of  peas  to  which  no 
nitrogen  was  added  most  of  the  plants  were  apparently  limited  in 
their  growth  by  the  amount  of  nitrogen  locked  up  in  the  seed. 
Here  and  there,  however,  a  plant,  under  apparently  the  same  cir- 
cumstances, grew  luxuriantly,  and  possessed  on  its  rootlets  abundant 
nodules.  The  experiments  of  Sir  John  Lawes  and  Sir  Henry  Gilbert 
at  Kothamsted*  demonstrated  further  that  under  the  influence  of 
suitable  microbe-seeding  of  the  soil  in  which  Leguminosse  were 
planted  there  is  nodule  formation  on  the  roots,  and  coincidentally 
increased  growth  and  gain  of  nitrogen  beyond  that  supplied  either 
in  the  soil  or  in  the  seed  as  combined  nitrogen.  Presumably  this  is 
due  to  the  fixation,  in  some  way,  of  free  nitrogen.  Nobbe  proved 
the  gain  of  nitrogen  by  non-leguminous  plants  (Elceagnus,  etc.)  when 
these  grow  root  nodules  containing  bacteria,  but  to  all  appearances 
bacteria  differing  morphologically  from  the  Bacillus  radicicola  of  the 
leguminous  plants. 

These  facts  being  established,  the  question  naturally  arises,  How 
is  the  fixation  of  nitrogen  to  be  explained,  and  by  what  species  of 
bacteria  is  it  performed  ?  In  the  first  place,  these  matters  are 
simplified  by  the  fact  that  there  is  very  little  fixation  indeed  by 
bacteria  in  the  soil  apart  from  symbiosis  with  higher  plants.  Hence 
we  have  to  deal  mainly  with  the  work  of  bacteria  in  the  higher 
plant.  Sir  Henry  Gilbert  concludes  f  that  the  alternative  explana- 
tions of  the  fixation  of  free  nitrogen  in  the  growth  of  Leguminosse 
seem  to  be: 

"  1.  That  under  the  conditions  of  symbiosis  the  plant  is  enabled 
to  fix  the  free  nitrogen  of  the  atmosphere  by  its  leaves ; 

"2.  That  the  nodule  organisms  become  distributed  within  the 
soil  and  there  fix  free  nitrogen,  the  resulting  nitrogenous  compounds 
becoming  valuable  as  a  source  of  nitrogen  to  the  roots  of  the  higher 
plant ; 

"3.  That  free  nitrogen  is  fixed  in  the  course  of  the  development 
of  the  organisms  within  the  nodules,  and  that  the  resulting  nitrogenous 
compounds  are  absorbed  and  utilised  by  the  host.  "  Certainly,"  he 
adds,  "the  balance  of  evidence  at  present  at  command  is  much  in 
favour  of  the  third  mode  of  explanation."  If  this  is  finally  proved 
to  be  the  case,  it  will  furnish  another  excellent  example  of  the  power 
existing  in  bacteria  of  assimilating  an  elementary  substance. 

Experiments  at  Eothamsted  have  confirmed  those  of  others,  in 
showing  that,  by  adding  to  a  sterilised  sandy-soil  growing  leguminous 

*  Sir   Henry  Gilbert,    F.R.S.,    The  Lawes  Agricultural   Trust  Lectures,  1893, 
p.  129. 

t  Ibid.,  p.  140. 


PLATE  12. 


NITROGEN-FIXING  BACTERIA  in  situ  IN  NODULE  ON  ROOTLET  OF  PEA. 
X    400. 


N"lTROGEN-FlXING   BACTERIA    in  SttU  IN    ROOT-NODULE 

OF  PEA.    (Section  of  Nodule),     x  500. 


NITROGEN-FIXING  BACTERIA  in  situ  IN  ROOT-NODULE 
OF  PEA.    (Section  of  Nodule),     x  600. 


[To  face  page  134. 


NITROGEN-FIXING  BACTERIA  135 

plants,  a  small  quantity  of  the  watery  extract  of  a  soil  containing 
the  appropriate  organisms,  a  marked  development  of  the  so-called 
leguminous  nodules  on  the  roots  is  induced,  and  that  there  is  coinci- 
dently  increased  growth,  and  gain  of  nitrogen.  There  is  no  evidence 
that  the  leguminous  plant  itself  assimilates  free  nitrogen ;  the  supposi- 
tion is,  that  the  gain  is  due  to  the  fixation  of  nitrogen  in  the  course  of 
development  of  the  lower  organisms  within  the  root-nodules,  the  nitro- 
genous compounds  so  produced  being  taken  up  and  utilised  by  the 
higher  plant. 

It  would  seem,  therefore,  that  in  the  growth  of  leguminous  crops, 
such  as  clover,  vetches,  peas,  beans,  sainfoin,  lucerne,  etc.,  at  any 
rate  some  of  the  large  amount  of  nitrogen  which  they  contain,  and  of 
the  large  amount  which  they  frequently  leave  as  nitrogenous  residue 
in  the  soil  for  future  crops,  may  be  due  to  atmospheric  nitrogen 
brought  into  combination  by  the  agency  of  lower  organisms.  It  has 
yet  to  be  ascertained,  however,  under  what  conditions  a  greater  or 
less  proportion  of  the  total  nitrogen  of  the  crop  will  be  derived — on 
the  one  hand  from  nitrogen-compounds  within  the  soil,  and  on  the 
other  from  such  fixation.  It  might  be  supposed,  that  the  amount 
due  to  fixation  would  be  the  less  in  the  richer  soils,  and  the  greater 
in  soils  that  are  poor  in  combined  nitrogen,  and  which  are  open  and 
porous.  On  the  other  hand,  recent  results  obtained  at  Eothamsted 
indicate  that,  at  any  rate  with  some  leguminous  plants,  there  may  be 
more  nodules  produced,  and  presumably  more  fixation,  with  a  soil 
rich  in  combined  nitrogen,  than  in  one  poor  in  that  respect. 

Most  authorities  would  agree  that  all  absorption  of  free  nitrogen, 
if  by  means  of  bacteria,  must  be  through  the  roots.  As  a  matter  of 
fact,  legumes,  especially  when  young,  use  nitrogen,  like  all  other 
plants,  derived  from  the  soil.  It  has  been  pointed  out  that,  unless 
the  soil  is  somewhat  poor  in  nitrogen,  there  appears  to  be  but  little 
assimilation  of  free  nitrogen  and  but  a  poor  development  of  root 
nodules.*  The  free  nitrogen  made  use  of  by  the  micro-organism  is 
in  the  air  contained  in  the  interstices  of  the  soil.  For  in  all  soils, 
but  especially  in  well-drained  and  light  soils,  there  is  a  large  quantity 
of  air.  Although  it  is  not  known  how  the  micro-organisms  in  legumes 
utilise  free  nitrogen  and  convert  it  into  organic  compounds  in  the 
tissues  of  the  rootlet  or  plant,  it  is  known  that  such  nitrogen  com- 
pounds pass  into  the  stem  and  leaves,  and  so  make  the  roots  really 
poorer  in  nitrogen  that  the  foliage.  But  the  ratio  is  a  fluctuating 
one,  depending  chiefly  on  the  stage  of  growth  or  maturity  of  the 
plant. 

If  the  nodules  from  the  rootlets  of  Leguminosse  be  examined,  the 
nitrogen-fixing  bacteria  can  be  readily  seen.  They  may  be  isolated 

*  This  has  been  denied  in  the  official  report  by  the  chemist  of  the  Experi- 
mental Farm  to  the  Minister  of  Agriculture  at  Ottawa  (Report,  1896,  p.  200). 


136  BACTERIA  IN  THE  SOIL 

and  grown  in  pure  culture  as  follows : — The  nodules  are  removed,  if 
possible,  at  an  early  stage  in  their  growth,  and  placed  for  a  few 
minutes  in  a  steam  steriliser.  This  is  advisable  in  order  to  remove 
the  various  extraneous  organisms  attached  to  the  outer  covering  of 
the  nodule.  The  latter  may  then  be  washed  in  antiseptic  solution, 
and  their  capsules  softened  by  soaking.  When  opened  with  a 
sterilised  knife,  thick  creamy  matter  exudes.  On  microscopic 
examination  this  is  found  to  be  densely  crowded  with  small  round- 
ended  bacilli  or  oval  bodies,  known  as  bacteroids.  By  a  simple 
process  of  hardening  and  using  the  microtome,  excellent  sections  of 
the  nodules  can  be  obtained  which  show  these  bacteria  in  situ.  In 
the  central  parts  of  the  section  may  be  seen  densely  crowded  colonies 
of  the  bacteria,  which  in  some  cases  invade  the  cellular  capsule  of 
the  nodule  derived  from  the  rootlet. 

The  organisms  are  of  various  shapes,  sometimes  rod-like,  and  at 
other  times  assuming  a  V  or  Y  shape.  Probably  these  latter  forms 
are  due  either  to  circumflex  arrangement,  branching  or  pleomorphism. 
At  the  end  of  the  summer  most  nodule-bearing  roots,  being  annuals, 
perish,  and  the  nitrogen-fixing  bacteria  are  liberated  in  the  surround- 
ing soil.  Probably  they  are  able  to  exist  for  long  periods  in  the  soil, 
and  re-infect  other  rootlets. 

Other  Bacterial  Symbioses. — As  we  have  already  pointed  out, 
incidental  association  of  organisms  must  not  be  mistaken  for  sym- 
bioses.  The  decomposition  organism,  B.  ramosus,  may  be  found 
associated  with  Nitrosomonas  and  Nitrdbacter  in  the  processes  of 
denitrification  and  nitrification,  but  this  does  not  necessarily  fulfil 
the  conditions  of  symbiosis,  even  though  each  of  the  three  produces 
substances  which  provide  pabulum  for  the  other  two.  True  symbiosis 
involves  a  much  closer  relationship  than  this,  namely,  the  inability 
of  each  symbiont  to  produce  its  effect  apart  from  its  partner. 

Now,  in  addition  to  the  case  of  the  nitrogen-fixing  bacteria  we 
have  other  bacterial  examples,  and  brief  reference  must  be  made  to 
them.  Van  Senus,  for  instance,  found  an  anaerobic  bacillus  capable  of 
dissolving  cellulose  if  associated  with  another  organism,  also  incapable 
by  itself  of  attacking  cellulose.  Winogradsky,  too,  found  that  a 
certain  anaerobic  organism  (Clostridium  Pasteurianum),  if  supplied 
with  abundance  of  dextrose  but  no  oxygen,  could  fix  atmospheric 
nitrogen.  This  capacity  was  found  to  be  due  to  the  organism  being 
surrounded  with  aerobic  bacteria  acting  in  partnership  with  it. 
Probably,  also,  the  bacteria  concerned  in  the  reduction  of  sulphates, 
and  the  oxidation  of  sulphuretted  hydrogen,  as  also  the  iron  bacteria, 
are  further  examples  of  symbiosis.  Kephir  and  the  so-called  ginger- 
beer  plant  must  also  be  named  in  the  same  category.  Kephir  is  a 
common  beverage  amongst  the  Caucasians.  The  "  Kephir  grains  "  are 
in  reality  composed  of  three  separate  organisms.  The  first  is  a  fila- 


OTHER  FORMS  OF  SYMBIOSIS  137 

men  tons  bacterium  forming  "  zooglea."  The  second  is  a  lactic-acid- 
producing  bacillus,  and  the  third  is  a  yeast.  By  these  agents  a 
fermentation  is  set  up  in  the  milk  of  cows,  goats,  or  sheep.  "  The 
yeast  and  the  bacteria,  either  jointly  or  separately,  split  up  the 
lactose  or  milk-sugar  into  two  other  sugars,  galactose  and  glucose. 
The  yeast  then  forms  alcohol  from  the  latter,  and  the  bacterium 
lactic  acid  from  the  former"  (Green).  This  filamentous  bacillus 
probably  affects  the  casein.  The  outline  is,  it  is  true,  only  the 
probable  course  of  action,  as  full  details  as  to  the  whole  function  of 
the  separate  factors  are  not  yet  known. 

The  gingerbeer  plant  is  the  agent  of  fermentation  in  the  so-called 
"stone  gingerbeer,"  and  is  composed  essentially  of  two  organisms, 
one  a  yeast,  Saccharomyces  pyriformis,  the  other  a  bacillus,  Bacterium 
vcrmiforme.  It  rarely  happens  that  these  two  forms  are  found  pure, 
there  being  as  a  rule  an  admixture  of  other  organisms  with  them. 
Professor  Green  describes  B.  vermiforme  as  growing  in  two  different 
ways,  namely,  as  long  rods  or  convoluted  threads,  invested  by  a 
translucent  wrinkled  sheath,  and  as  constituent  microbes  contained 
within  the  sheath,  yet  able  to  escape  from  it.  The  sheathing  form 
of  the  organism  can  only  be  produced  when  oxygen  is  replaced  by 
carbon  dioxide.  In  the  symbiotic  association  the  yeast  absorbs  the 
oxygen,  and  during  its  fermentative  activity  produces  carbon  dioxide, 
thus  providing  the  necessary  conditions  for  the  formation  of  the 
sheath.  The  bacterium  benefits  by  substances  excreted  by  the  yeast, 
and  the  latter  profits  in  its  turn  by  the  removal  of  these  matters 
through  the  agency  of  the  former.  The  yeast  sets  up  the  usual 
fermentation  of  cane-sugar. 

A  third  organism  manifesting  symbiosis  occurs  in  Madagascar 
as  a  curious  gelatinous  substance  found  attacking  the  sugar-cane, 
and  consisting  again  of  a  yeast  and  a  bacterium  associated  together 
in  very  much  the  same  way  as  are  the  organisms  in  the  gingerbeer 
ferment. 

Before  leaving  this  subject  of  symbiosis  as  illustrated  in  the 
lichens,  in  Winogradsky's  Clostridium,  in  the  nodule-bacteria,  in  the 
gingerbeer  plant,  and  in  Kephir,  we  may  suitably  inquire  whether 
anything  is  at  present  known  as  to  how  the  symbionts  are  related  to 
each  other.  Obviously  the  matter  presents  many  difficulties,  and 
the  problem  is  by  no  means  solved.  There  are,  however,  three  chief 
hypotheses.  First,  the  provision  of  definite  food  materials  by  the 
one  symbiont  for  the  other  may  be  an  important  factor ;  e.g.  an  alga 
supplies  a  fungus  with  carbo-hydrates,  or  a  fungus  converts  starch 
into  the  fermentable  sugars  which  the  associated  yeast  needs.  In 
other  cases  the  advantage  derived  is  one  of  protection  from  some 
injurious  agent;  e.g.,  the  aerobic  bacterium  prevents  the  access  of 
oxygen  to  the  anaerobic  one.  Thirdly,  there  is  some  evidence, 


138  BACTERIA  IN  THE  SOIL 

according  to  Professor  Marshall  Ward,  in  support  of  the  hypothesis 
that  one  symbiont  may  stimulate  another  by  exciting  some  body 
which  acts  as  an  exciting  drug  to  the  latter — just  as  truly  as  certain 
drugs  act  as  stimulants  to  some  cell  or  organ  of  a  higher  animal,  and 
probably  in  a  fundamentally  similar  manner. 

Before  we  leave  the  subject  of  the  economic  bacteria  present  in 
the  soil,  it  may  be  well  to  refer  briefly  to  the  application  of  the  new 
knowledge  to  agriculture.  Whilst  many  of  the  details  of  our  know- 
ledge concerning  "the  living  earth"  have  not  passed  beyond  the 
experimental  stage,  it  is  not  to  be  wondered  at  that  the  New  Soil 
Science  has  been  received  with  some  caution,  and  possibly  in  some 
quarters  even  scepticism.  This  is  neither  surprising,  nor,  as  regards 
the  details,  altogether  undesirable.  A  number  of  the  cardinal 
principles,  however,  are  now  obtaining  very  general  acceptance 
amongst  practical  agriculturists.  Briefly,  these  may  be  stated  as 
follows.  That  a  soil  which  has  been  sterilised,  or  is  otherwise  not 
occupied  by  soil  bacteria,  is  necessarily  an  unfertile  soil ;  that  the 
disintegration  and  oxidation  of  organic  matter  in  the  soil  are  the 
result  of  bacterial  life  and  work;  that  the  sowing,  growing,  and 
feeding  of  the  desirable  soil  germs  are  of  as  much  importance  to  the 
agriculturist  to-day  as  is  the  sowing  of  seeds,  or  the  growing  and 
feeding — by  manuring — of  plants ;  that  the  physical  and  chemical 
conditions  of  soil  favourable  to  these  bacteria  are  of  as  much  value 
to  the  agriculturist  as  the  requisite  physical  and  chemical  conditions 
for  the  growth  of  the  yeast  cell  are  to  the  brewer ;  and  indeed  that 
one  of  the  essential  functions  of  manure  in  the  soil  is  to  provide 
favourable  pabulum  and  conditions  for  the  operation  of  these  soil 
ferments. 

In  the  further  elucidation  of  these  principles  various  series  of 
experiments,  in  addition  to  those  at  Eothamsted  (under  Sir  J.  B. 
Lawes  and  Sir  J.  H.  Gilbert)  and  at  Woburn  (under  Dr  Voelcker), 
have  been  designed  and  carried  out.  Of  such  a  nature  are  the  well- 
known  Dalmeny  Experiments  originated  by  Lord  Eosebery  some 
years  ago.  The  chemists  engaged  in  this  series  were  aware  that 
though  large  doses  of  caustic  lime  would  kill  outright  certain  of  the 
economic  soil  bacteria,  annual  or  biennial  dressings  of  mild  lime 
added  to  the  culture  media,  that  is  the  soil,  would  materially  assist. 
the  process  of  nitrification.  Five  acres  of  land  have  been  worked  as 
a  miniature  farm,  each  division  being  divided  into  sixteen  plots.  The 
soil  is  of  very  uniform  character,  and  is  of  the  usual  loamy  kind  met 
with  in  the  low  grounds  of  the  Lothians.  Each  plot  has  been 
manured,  or  left  unmanured  as  the  case  may  be,  on  a  regular  system, 
so  that  the  residual  values  of  the  different  manures,  as  well  as  the 
yield  of  crop,  may  be  accurately  dealt  with.  The  crops  grown  are 
regularly  analysed  in  order  to  determine  the  feeding  value.  Concern- 


SAPROPHYTES  139 

ing  results,  it  may  be  said  that  the  wheat  plot  points  to  the  fact  that 
where  the  soil  is  in  good  condition,  through  the  application  of  farm- 
yard manure,  the  artificials  that  may  be  most  profitably  applied  are 
lime  (4  parts),  superphosphate  (3  parts),  and  sulphate  of  ammonia 
(1  part).  On  the  other  hand,  where  the  land  is  not  in  such  high 
condition,  this  dressing  should  be  supplemented  by  a  dressing  of 
potash  salt.  The  analyses  show  that  by  the  application  of  these 
dressings  the  value  and  quality  of  the  crop  are  increased  because 
the  operations  of  the  nitrifying  organisms  have  been  thus  favoured. 

4.  The  Saprophytie  Bacteria  in  Soil 

This  group  of  micro-organisms  is  by  far  the  most  abundant 
as  regards  number.  They  live  on  the  dead  organic  matter  of  the 
soil,  and  their  function  appears  to  be  to  break  it  down  into  simpler 
constitution.  Specialisation  is  probably  progressing  among  them, 
for  their  name  is  legion,  and  the  struggle  for  existence  keen.  After 
we  have  eliminated  the  economic  bacteria,  most  of  which  are  obviously 
saprophytes,  the  group  is  greatly  reduced.  It  is  also  needless  to  add 
that  of  the  remnant  little  beyond  morphology  is  known,  for  as  their 
function  is  learned  they  are  classified  otherwise.  It  is  probable,  as 
suggested,  that  many  of  the  species  of  common  saprophytes  normally 
existent  in  the  soil  act  as  auxiliary  agents  to  denitrification  and 
putrefaction.  At  present  we  fear  they  are  disregarded  in  equal 
measure,  and  for  the  same  reasons,  as  the  common  water  bacteria. 
An  excess  of  either,  in  soil  or  water,  is  not  of  itself  injurious  as  far 
as  we  know ;  indeed,  it  is  probably  just  the  reverse.  It  is,  however, 
frequently  an  index  of  value  as  to  the  amount  and  sometimes  con- 
dition of  the  contained  organic  matter.  The  remarks  made  when 
considering  water  bacteria  apply  here  also,  viz.,  that  an  excess  of 
saprophytes  acts  not  only  as  index  of  increase  of  organic  matter, 
but  as  at  first  auxiliary,  and  then  detrimental,  to  pathogenic  organisms. 
It  will  require  accurate  knowledge  of  soil  bacteria  generally  to  be 
able  to  say  which  saprophytic  germs,  if  any,  have  no  definite  function 
beyond  their  own  existence.  It  may  be  doubted  whether  the  stern 
behests  of  nature  permit  of  such  organisms.  However  that  may  be, 
we  may  feel  confident,  though  at  present  there  are  many  common 
bacteria  in  soil,  as  also  in  water,  the  life  object  of  which  is  not 
ascertained,  that  as  knowledge  increases  and  becomes  more  accurate, 
this  special  provisional  group  will  become  gradually  absorbed  into 
other  groups  having  a  part  in  the  economy  of  nature,  or  in  the 
production  of  disease.  At  present  the  decomposition,  denitrifying, 
nitrifying,*  and  nitrogen-fixing  organisms  are  the  only  saprophytes 

*  It  has  already  been  pointed  out  that  the  nitrifying  bacteria,  though  able  to 
live  on  organic  matter,  do  not  require  such  either  for  existence  or  for  the  performance 
of  their  function. 


140  BACTERIA  IN  THE  SOIL 

which  have  been  rescued  from  the  oblivion  of  ages,  and  brought  more 
or  less  into  daylight.  It  is  but  our  lack  of  knowledge  which  requires 
the  present  division  of  saprophytes,  whose  business  and  place  in 
the  world  is  unknown. 


5.  The  Pathogenic  Organisms  found  in  Soil 

In  addition  to  these  saprophytes  and  the  economic  bacteria, 
there  are,  as  is  now  well  known,  some  disease-producing  bacteria 
finding  their  nidus  in  ordinary  soil.  The  three  chief  members  of  this 
group  are  the  bacillus  of  Tetanus,  the  bacillus  of  Quarter-Evil,  and 
the  bacillus  of  Malignant  (Edema. 

Tetanus 

The  pathology  of  this  disease  has,  during  recent  years,  been 
considerably  elucidated.  It  was  the  custom  to  look  upon  it  as 
"  spontaneous,"  and  arising  no  one  knew  how ;  now,  however,  after 
the  experiments  of  Sternberg  and  Mcolaier,  the  disease  is  known  to 
be  due  to  a  micro-organism  common  in  the  soil  of  certain  localities, 
existing  there  either  as  a  bacillus  or  in  a  resting  stage  of  spores. 
Fortunately,  Tetanus  is  comparatively  rare,  and  one  of  the  peculiar 
biological  characters  of  the  bacillus  is  that  it  only  grows  in  the 
absence  of  oxygen.  This  fact  contributed  not  a  little  to  the  difficulties 
which  were  met  with  in  securing  its  isolation. 

Tetanus  occurs  in  man  and  horses  most  commonly,  though  it  may 
affect  other  animals.  There  is  usually  a  wound,  often  an  insignificant 
one,  which  may  occur  in  any  part  of  the  body.  The  popular  idea 
that  a  severe  cut  between  the  thumb  and  the  index  finger  leads  to 
tetanus  is  without  scientific  foundation.  As  a  matter  of  fact,  the 
wound  is  nearly  always  on  one  or  other  of  the  limbs,  and  becomes 
infected  simply  because  the  limbs  come  more  into  contact  with  soil 
and  dust  than  does  the  trunk.  It  is  not  the  locality  of  the  wound 
nor  its  size  that  affects  the  disease.  A  cut  with  a  dirty  knife,  a  gash 
in  the  foot  from  the  prong  of  a  gardener's  fork,  the  bite  of  an  insect, 
or  even  the  prick  of  a  thorn,  have  before  now  set  up  tetanus. 
Wounds  which  are  jagged,  and  occurring  in  absorptive  tissues,  are 
those  most  fitted  to  allow  the  entrance  of  the  bacillus.  The  wound 
forms  a  local  factory,  so  to  speak,  of  the  bacillus  and  its  secreted 
poisons ;  the  bacillus  always  remains  in  the  wound,  but  the  toxins 
may  pass  throughout  the  body,  and  are  especially  absorbed  by  the 
cells  of  the  central  nervous  system,  and  thus  give  rise  to  the  spasms 
which  characterise  the  disease.  Suppuration  generally  occurs  in  the 
wound,  and  in  the  pus  thus  produced  may  be  found  a  great  variety 
of  bacteria,  as  well  as  the  specific  agent  itself.  After  a  few  days  or, 


PLATE  13. 


Bacillus  tetani. 

preparation,  from  broth  culture,  showing  spore 
formation,     x   1000. 


Bacillus  mycoides. 

Film  preparation,  from  agar  culture,  37°  C.    Spore 
formation,     x  1000. 


Streptothrix  actinomyces. 

ay  fungus  in  tissue.    Stained  by  Gram's  method. 
x   700. 


Bacillus  mallei  (Glanders). 

Film  preparation,  agar  culture,  37*  C. 
X   1000. 


[To  face  page  140, 


TETANUS  141 

it  may  be,  as  much  as  a  fortnight,  when  the  primary  wound  may  be 
almost  forgotten,  general  symptoms  occur.  Their  appearance  is  often 
the  first  sign  of  the  disease.  Stiffness  of  the  neck  and  facial  muscles, 
including  the  muscles  of  the  jaw,  is  the  most  prominent  sign.  This 
is  rapidly  followed  by  spasms  and  local  convulsions,  which,  when 
affecting  the  respiratory  or  alimentary  tract,  may  cause  a  fatal  result. 
Fever  and  increased  rate  of  pulse  and  respiration  are  further  signs 
of  the  disease  becoming  general.  After  death,  which  results  in  the 
majority  of  cases,  there  is  very  little  to  show  the  cause  of  fatality. 
The  wound  is  observable,  and  patches  of  congestion  may  be  found 
on  different  parts  of  the  nervous  system,  particularly  the  medulla 
(grey  matter),  pons,  and  even  cerebellum.  Evidence  has  recently 
been  forthcoming  at  the  Pasteur  Institute  to  support  the  theory 
that  tetanus  is  a  "nervous"  disease,  more  or  less  allied  to  rabies, 
and  is  best  treated  by  intra-cerebral  injection  of  antitoxin,  which 
then  has  an  opportunity  of  opposing  the  toxins  at  their  favourite 
site.  The  toxins  diffuse  throughout  the  tissues  of  the  body,  but 
particularly  affect  the  spinal  cord.  The  long  incubation  period 
indicates  that  the  toxins  are  probably  produced  by  a  ferment  of 
some  kind.  Whatever  its  exact  nature,  it  is  undoubtedly  a  most 
powerful  poison. 

Tetanus  bacilli  spores  have  been  found  in  considerable  quantities 
in  the  dust  of  dry  jute  fibre  ( Andre wes),  and  various  cases  are  on 
record  where  the  disease  has  been  contracted  by  workers  in  jute 
mills  in  Dundee  and  elsewhere.  Legge  attributes  the  presence  of 
the  bacilli  in  the  jute  (Cor chorus)  to  the  soil  in  which  it  is  grown  in 
Bengal. 

The  Bacillus  of  Tetanus. — In  the  wound  the  bacillus  is  present 
in  large  numbers,  but  mixed  up  with  a  great  variety  of  suppurative 
bacteria  and  extraneous  organisms.  It  is  in  the  form  of  a  straight 
short  rod  with  rounded  ends,  occurring  singly  or  in  pairs  or  threads, 
and  slightly  motile.  It  has  been  pointed  out  that  by  special  methods 
of  staining  flagella  may  be  demonstrated.  These  are  both  lateral 
and  terminal,  thin  and  thick,  and  are  shed  previously  to  sporulation. 
Branching  also  has  been  described.  Indeed,  it  would  appear  that, 
like  the  bacillus  of  tubercle,  this  organism  has  various  polymorphic 
forms.  Next  to  the  ordinary  bacillus,  filamentous  forms  predominate, 
particularly  so  in  old  cultures.  Clubbed  forms,  not  unlike  the 
bacillus  of  diphtheria,  may  often  be  obtained  from  agar  cultures. 
Without  doubt  the  most  peculiar  characteristic  of  this  bacillus  is  its 
sporulation.  The  well-formed  round  spores  occur  readily  at  incuba- 
tion temperature.  They  occupy  a  position  at  one  or  other  pole  of 
the  bacillus,  and  have  a  diameter  considerably  greater  than  the 
organism  itself.  Thus  the  well-known  "  drumstick  "  form  is  produced. 
In  practice  the  spores  frequently  occur  free  in  the  medium  and  in 


142  BACTERIA  IN  THE  SOIL 

microscopical  preparation.  Like  other  spores,  they  are  extremely 
resistant  to  heat,  desiccation,  and  antiseptics.* 

As  we  have  seen,  this  bacillus  is  a  strict  anaerobe,  growing  only 
in  the  absence  of  oxygen.  The  favourable  temperature  is  37  °  C.,  and 
it  will  only  grow  very  slowly  at  or  below  room  temperature.  The 
organism  is  readily  stained  by  the  ordinary  stains  and  by  Gram's 
method. 

An  excellent  culture  is  generally  obtainable  in  glucose  gelatine. 
The  growth  occurs  only  in  the  depth  of  the  medium,  and  appears  as 
fine  threads  passing  horizontally  outwards  from  the  track  of  the 
needle.  At  the  top  and  bottom  of  the  growth  these  fibrils  are 
shorter  than  at  the  middle  or  somewhat  below  the  middle.  For 
extraction  of  the  soluble  products  of  the  bacillus,  glucose  broth  may 
be  used.  (For  isolation  and  detection  of  the  B.  tetani,  see  Appendix, 
p.  481.) 

In  some  countries,  and  in  certain  localities,  the  bacillus  of  tetanus 
is  a  very  common  habitant  of  the  soil,  and  when  one  thinks  how 
frequently  wounds  must  be  more  or  less  contaminated  with  such 
soil,  the  question  naturally  arises,  How  is  it  that  the  disease  is, 
fortunately,  so  rare?  Probably  we  must  look  to  the  advance  of 
bacteriological  science  to  answer  this  and  similar  questions  at  all 
adequately.  Much  has  recently  been  done  in  Paris  and  elsewhere  to 
emphasise  the  relation  which  other  organisms  have  to  such  bacteria 
as  those  of  typhoid  and  tetanus.  In  tetanus,  Kitasato,  Vaillard,  and 
others  have  pointed  out  that  the  presence  of  certain  other  bacteria, 
or  of  some  foreign  body,  is  necessary  to  the  production  of  the  disease. 
The  common  organisms  of  suppuration  in  particular  appear  to 
increase  the  virulence  of  the  bacillus  of  tetanus.  How  these 
auxiliary  organisms  perform  this  function  has  not  been  fully 
elucidated.  Probably,  however,  it  is  by  damaging  the  tissues  and 
weakening  their  resistance  to  such  a  degree  as  to  afford  a  favourable 
multiplying  ground  for  the  tetanus  bacillus.  Some  authorities  hold 
that  they  act  by  using  up  the  surrounding  oxygen,  and  so  favouring 
the  growth  of  the  germ  of  tetanus.  In  any  case  it  is  now  generally 
held  that  in  natural  infection  the  presence  of  some  foreign  body  or 
suppurative  bacteria  is  necessary  to  produce  the  disease. 

Quarter-Evil  and  Malignant  (Edema 

Quarter-Evil  (or  symptomatic  anthrax)  is  a  disease  of  animals, 
produced  in  a  manner  analogous  to  tetanus.  It  is  characterised  by 
a  rapidly-increasing  swelling  of  the  upper  parts  of  the  thighs,  sacrum, 
etc.,  which,  beginning  locally,  may  attain  to  extraordinary  size  and 

*  Atlas  and  Principles  of  Bacteriology,  by  Lehman n  and  Neumann,  part  ii., 
pp.  330-337. 


QUARTER-EVIL  143 

extent.  The  swelling  may  assume  a  dark  colour,  and  crackles  on 
being  touched.  There  is  high  temperature,  and  secondary  motor  and 
functional  disturbances.  The  disease  ends  fatally  in  two  or  three 
days. 

Slight  injuries  to  the  surface  of  the  skin  or  mucous  membrane 
are   sufficient   for   the   introduction   of    the 
causal  bacillus.     This  organism  is,  like  the         ^         \       ° 
bacillus  of  tetanus,  an  anaerobe,  existing  in  ^      5-        \ 

the  superficial  layers  of  the  soil.     From  its        .  I   '*  A    f     1 

habitat  it  readily  gains  entrance  to  animal       /      /  § 

tissues.     It    has    spores,   but   though    they         Q  V 

are   of   greater   diameter   than   the  bacillus     i      A    J          \     | 
itself,    they    are    not    absolutely    terminal.  1*0 

Hence    they    merely    swell    out    the    cap-  * 

sule   of   the   bacillus,  and  produce  a   club-  S      1 

shaped  rod.     The  bacillus  forms  gas  while 

growing  in  the  tissues^  and  in  artificial  cul-      pio  20._Diagram  of  Bacillus 
ture.      External    physical    conditions    have         of  symptomatic  Anthrax. 
little   effect  upon   this  organism,  and  dried 

and  even  buried  flesh  retains  infection  for  a  long  period  of 
time. 

Quarter-Ill,  Quarter-Evil,  or  Black-Leg 

Quarter-ill  may  be  said  to  lack  much  of  the  importance  and  interest  which  is 
attached  to  anthrax,  inasmuch  as  it  is  confined  to  two  domestic  animals — sheep  and 
cattle — and  is  not  communicable  to  man.  It,  however,  resembles  anthrax,  in  so 
far  as  they  are  both  caused  by  the  introduction  into  the  blood  of  the  healthy 
animal  of  specific  bacilli.  Both  diseases  have  a  tendency  to  recur  on  farms  or 
premises  on,  or  in,  which  animals  affected  with  these  diseases  have  been  previously 
kept.  On  the  other  hand  neither  anthrax  nor  quarter-ill  is  communicable  by 
association  of  the  affected  with  the  healthy  animal,  and  in  that  respect  they  differ 
from  most  of  the  contagious  diseases  which  are  legislated  for  in  this  country. 
Another  peculiar  feature  of  quarter-ill  is  that  while  it  is  very  fatal  to  sheep  at  any 
age,  cattle  over  two  years  may  be  said  to  have  an  immunity  against  the  disease. 

The  symptoms  of  quarter-ill  in  young  cattle  are  so  strikingly  different  from  any 
other  disease  that  an  error  in  diagnosis  is  almost  impossible.  The  first  indication  of 
an  animal  being  affected  with  quarter-ill  is  a  marked  stiffness  or  lameness  of  one  of 
the  limbs ;  it  is  exceedingly  dull,  and  presents  a  most  anxious  and  dejected 
appearance,  does  not  feed,  and  it  is  with  extreme  difficulty  that  it  can  be  forced  to 
move.  Very  soon  after  the  limb  is  attacked  a  swelling  appears  beneath  the  skin, 
usually  upon  one  of  the  hind  quarters,  which  is  extremely  hot,  increases  in  size 
rapidly,  and  is  most  painful  to  the  animal  when  touched.  This  swelling  has  a 
disposition  to  extend  down  the  leg,  or  perhaps  along  the  loins  and  back,  and  when 
pressed  gives  a  peculiar  crackling  sensation  to  the  fingers.  In  almost  every  instance 
death  supervenes  within  a  few  hours  after  the  swelling  has  appeared. 

In  the  case  of  sheep  the  symptoms  are  not  of  so  marked  a  character.  The  first 
indication  is  lameness,  but -the  swelling  is  not  so  observable  in  sheep  as  in  cattle, 
being  hidden  to  a  great  extent  in  the  case  of  the  former  by  the  fleece. 

There  is  no  doubt  that  the  disease  exists  to  a  greater  extent  among  the  sheep  in 
certain  counties  in  England  than  has  been  generally  known,  and  from  the  rapidity 
with  which  sheep  frequently  die  it  is  often  locally  called  **  strike." 

Should  any  doubt  exist  as  to  whether  a  sheep  has  died  from  quarter-ill,  the 
difficulty  can  easily  be  solved  by  making  an  incision  through  the  skin  of  the  dead 


144  BACTERIA  IN  THE  SOIL 

animal  into  the  tumour  or  swelling,  which  contains  a  large  quantity  of  dark 
coloured  fluid,  which  emits  a  very  strong  and  peculiarly  offensive  odour.  Any 
fluid  that  may  thus  escape  should  be  carefully  collected  and  destroyed. 

The  carcases  of  animals  which  have  died  of  quarter-ill  should  be  buried  as  in 
anthrax,  or,  still  better,  cremated  on  or  in  the  place  where  the  animal  died.  All 
dung,  fodder,  litter,  or  other  materials  of  a  like  character  which  may  have  been 
on  or  about  places  or  sheds  where  animals  have  died  should  be  burnt,  or  thoroughly 
mixed  with  some  powerful  disinfectant,  and  buried  in  a  part  of  the  premises  to 
which  cattle  and  sheep  do  not  have  access.  The  sheds,  particularly  the  flooring 
and  mangers,  should  be  thoroughly  washed  and  scrubbed  with  a  5  per  cent,  solution 
of  carbolic  acid,  and  it  would  be  prudent  to  repeat  the  process  before  they  are 
again  used  for  cattle  or  sheep. 

A  third  disease-producing  microbe  found  naturally  in  soil  is  that 
which  produces  the  disease  known  as  Malignant  (Edema.  Pasteur 
called  this  disease  gangrenous  septicaemia,  and  the  bacillus  vibrion 
septique.  Unlike  quarter-evil,  malignant  oedema  may  occur  in  man 
in  cases  where  wounds  have  become  septic.  It  is  usually  described 
as  a  spreading  inflammatory  oedema,  with  emphysema,  and  followed 
by  gangrene.  Man  and  animals  become  inoculated  with  this  bacillus 
from  the  surface  of  soil,  straw-dust,  upper  layers  of  garden-earth,  or 
decomposing  animal  and  vegetable  matter. 

The  bacillus  occurs  in  the  blood  and  tissues  as  a  long  thread 
(3  JUL  to  10  /UL  in  length),  composed  of  slender  segments  of  irregular 
length.  It  is  motile  and  anaerobic,  and  readily  stained  by  aniline 
stains  but  not  by  Gram's  method,  in  this  way  differing  from  the 
anthrax  bacillus.  The  spores  are  larger  than  the  diameter  of  the 
bacillus,  and  usually  centrally  placed.  The  organism  produces  gas, 
and  so  much  is  this  the  case  in  artificial  culture,  that  the  medium 
itself  is  frequently  split  up.  The  bacillus  liquefies  gelatine.  The 
most  suitable  medium  for  cultivation  is  glucose  agar  at  37°  G. 

Both  malignant  oedema  and  symptomatic  anthrax  are  similar  in 
some  respects  to  anthrax  itself.  There  are,  however,  a  number  of 
points  for  differential  diagnosis.  The  enlargement  of  the  spleen,  the 
enormous  numbers  of  bacilli  throughout  the  body,  the  square  ends 
of  the  bacillus,  its  non-mo tility,  its  equal  inter- bacillary  spaces,  its 
aerobic  growth,  and  its  characteristic  staining,  afford  ample  evidence 
of  the  anthrax  bacillus. 

Frankel  and  Pasteur  have  both  demonstrated  the  possible  presence 
in  soil  of  the  bacillus  of  anthrax  itself.  Frankel  maintained  that  it 
could  not  live  there  long,  and  at  10  feet  below  the  surface  no  growth 
occurred.  This  may  have  been  due  to  the  low  temperature  of  such 
a  depth.  Pasteur  held  that  earthworms  are  responsible  for  convey- 
ing the  spores  of  anthrax  from  buried  carcases  to  the  surface,  and 
thus  bringing  about  re-infection.  In  any  case  it  is  well-known  that 
the  spores  of  anthrax  may  infect  a  soil  for  months.  The  bacillus  of 
cholera,  too,  has  been  successfully  grown  in  soil,  except  during 
winter.  The  presence  of  common  saprophytes  in  the  soil  is  prejudicial 


RELATION  TO  DISEASE  145 

to  the  development  of  the  cholera  spirillum,  and  under  ordinary 
circumstances  it  succumbs  in  the  struggle  for  existence.  Other 
species  of  bacteria  have  also  been  isolated  from  soil  from  time  to 
time. 

Now  whilst  since  the  early  days  of  bacteriology  the  three  organ- 
isms we  have  described  have  been  looked  upon  as  the  typical  bacteria 
of  soil,  modern  research  has  brought  to  light  a  new  relationship 
between  soil  and  disease,  which  has  greatly  enhanced  the  importance 
of  our  knowledge  of  the  subject.  Directly,  it  has  been  shown  that 
soil  may  harbour  germs  of  disease,  acting  sometimes  as  a  favourable 
and  at  other  times  as  an  unfavourable  nidus.  Indirectly,  it  has  been 
shown  that  a  right  understanding  of  the  bacteriology  of  water  arid 
its  potentiality  of  disease  production,  depends  upon  a  knowledge  of 
bacteria  in  the  soil  over,  or  through  which,  the  water  has  passed. 
The  matter  must,  therefore,  be  briefly  considered  here. 


The  Relation  of  Soil  generally  to  certain  Bacterial  Diseases 

It  is  now  some  years  since  Sir  George  Buchanan,  for  the  English 
Local  Government  Board,  and  Dr  Bowditch,  for  the  United  States, 
formulated  the  view  that  there  is  an  intimate  relationship  between 
dampness  of  soil  and  the  bacterial  disease  of  Consumption  (tuber- 
culosis of  the  lungs).  The  matter  was  left  at  that  time  sub  judicc, 
but  the  conclusion  has  since  been  drawn,  and  it  is  surely  a  legitimate 
one,  that  the  dampness  of  the  soil  acted  injuriously  in  one  of  two 
ways.  It  either  lowered  the  vitality  of  the  tissues  of  the  individual, 
and  so  increased  his  susceptibility  to  the  disease,  or  in  some  unknown 
way  favoured  the  life  and  virulence  of  the  bacillus.  That  is  one  fact. 
Secondly,  Pettenkofer  traced  a  definite  relationship  between  the  rise 
and  fall  of  the  ground  water  with  pollution  of  the  soil  and  enteric 
(typhoid)  fever.*  A  third  series  of  investigations  concluded  in  the 
same  direction,  viz.,  the  researches  by  Dr  Ballard  respecting  summer 
diarrhoea.  This,  it  is  generally  held,  is  a  bacterial  disease,  although 
no  single  specific  germ  has  been  isolated  as  its  cause.  Ballard 
demonstrated  that  the  summer  rise  of  diarrhoea  mortality  does  not 
commence  until  the  mean  temperature  of  the  soil,  recorded  by  the 
4-foot  thermometer,  has  attained  56 '4°  F.,  and  the  decline  of  such 
diarrhoea  coincides  more  or  less  precisely  with  the  fall  in  soil 
temperature.  This  temperature  (56*4°  F.)  is,  therefore,  considered 

*  The  conditions  requisite  for  an  outbreak  of  enteric  fever  were,  according  to 
Pettenkofer,  (a)  a  rapid  fall  (after  a  rise)  in  the  ground  water,  (6)  pollution  of  the 
soil  with  animal  impurities,  (c)  a  certain  earth  temperature,  and  lastly  (d)  a  specific 
micro-organism  in  the  soil.  These  four  conditions  have  not,  particularly  in  England, 
always  been  fulfilled  preparatory  to  an  epidemic  of  typhoid.  Yet  the  observations 
necessary  for  these  deductions  were  a  definite  step  in  advance  of  the  idea  of  the 
significance  of  mere  dampness  of  soil. 

K 


146  BACTERIA  IN  THE  SOIL 

as  the  "  critical "  4-foot  earth  temperature,  that  is  to  say,  the 
temperature  at  which  certain  changes  (putrefactive,  bacterial,  etc.) 
take  place  either,  primarily,  in  the  earth,  or  secondarily,  in  the  atmo- 
sphere, with  the  consequent  development  of  the  diarrhoeal  poison. 

After  prolonged  investigation  on  behalf  of  the  Local  Govern- 
ment Board,  Dr  Ballard  formulated  the  causes  of  diarrhoea  in  the 
following  conclusions : — * 

(a)  The  essential  cause  of  diarrhoea  resides  ordinarily  in  the  super- 
ficial layers  of  the  earth,  where  it  is  intimately  associated  with  the 
life  processes  of  some  micro-organism  not  yet  detected  or  isolated. 

(b)  That  the  vital  manifestations  of  such  organism  are  dependent, 
among  other  things,  perhaps  principally  upon  conditions  of  season 
and  the  presence  of  dead  organic  matter,  which  is  its  pabulum. 

(c)  That  on  occasion  such  micro-organism  is  capable  of  getting 
abroad  from  its  primary  habitat,  the  earth,  and  having  become  air- 
borne,  obtains    opportunity    for    fastening    on   non-living   organic 
material,  and  of  using  such  organic  matter  both  as  nidus  and  as 
pabulum  in  undergoing  various  phases  of  its  life-history. 

(d)  That  from  food,   as  also  from  contained  organic  matter  of 
particular    soils,    such    micro-organism    can    manufacture,    by   the 
chemical    changes   wrought    therein    through    certain    of    its    life 
processes,  a  substance  which  is  a  virulent  chemical  poison. 

Here,  then,  we  have  a  large  mass  of  evidence  from  the  data 
collected  by  Buchanan,  Bowditch,  Pettenkofer,  and  Ballard.  But 
much  of  this  work  was  done  anterior  to  the  time  of  the  application 
of  bacteriology  to  soil  constitution.  Recently  the  matter  has 
received  increased  attention  from  various  workers  abroad,  and  in  this 
country  from  Robertson,  Martin,  Houston,  and  others,  and  we  must 
now  consider  the  new  facts  brought  forward  by  these  investigators. 

From  the  first,  experiments  on  this  subject  have  been  more  or 
less  confined  to  the  behaviour  of  the  typhoid  bacillus  than  any  other 
pathogenic  organism.  This  has  been  partly  due  to  the  importance 
of  this  organism  in  relation  to  soil,  and  partly  because  it  is  more 
convenient  than,  say,  the  tubercle  bacillus  for  experimental  work.  In 
1888  G-rancher  and  Deschamps  showed  that  the  typhoid  bacillus  was 
able  to  survive  in  soil  for  more  than  twenty  weeks,f  and  Karlinski 
arrived  at  a  similar  conclusion.  J  In  1894  Dempster  published  the 
results  of  his  work  on  the  same  subject,  in  which  he  obtained  the 
typhoid  bacillus  from  sand  after  twenty-three  days,  from  garden  soil 
after  forty-two  days,  and  from  peat  not  later  than  twenty-four  hours.  § 
Four  years  later  came  Dr  Robertson's  valuable  researches  into  the 

*  Supplement  to  the  Report  of  the  Medical  Officer  of  the  Local  Government  Board, 
1887,  p.  7. 

t  Arch,  de  Med.  Exp.  et  d'Anat.  Path.,  1889,  7th  January. 
J  Arch.  f.  Hyg.,  Bd.  xiii.,  Heft  3. 
§  Brit.  Med.' Jour.,  1894,  i.,  p.  1126. 


TYPHOID  AND  SOIL  147 

growth  of  the  bacillus  of  typhoid  in  soil  of  an  ordinary  field.  By 
experimental  inoculation  of  the  soil  with  broth  cultures,  he  was  able 
to  isolate  the  bacillus  twelve  months  after,  alive  and  virulent.  He 
concluded  that  the  typhoid  organism  is  capable  of  growing  very 
rapidly  in  certain  soils,  and  under  certain  circumstances  can  survive 
from  one  summer  to  another.  The  rains  of  spring  and  autumn,  or 
the  frosts  and  snows  of  winter,  do  not  kill  it  off  so  long  as  there  is 
sufficient  organic  pabulum.  Sunlight,  the  bactericidal  power  of 
which  is  well  known,  had,  as  would  be  expected,  no  effect  except 
upon  the  bacteria  directly  exposed  to  its  rays.  The  bacillus  typhosus 
quickly  died  out  in  the  soil  of  grass-covered  areas.* 

Next  came  the  experiments  of  Dr  -Sidney  Martin,  which  were 
undertaken  to  inquire  into  the  extra-corporeal  existence  of  the 
bacillus  of  typhoid  fever  in  soil.  He  found,  after  a  prolonged 
research,  that  certain  cultivated  soils,  especially  garden  soils,  when 
sterilised  are  favourable  to  the  vitality  and  growth  of  this  bacillus, 
whether  the  soil  was  kept  at  room  temperature  (19°  C.)  or  blood-heat 
(37°  C.).  In  such  soils  the  B.  typhosus  was  still  alive  after  four 
hundred  and  four  days,  and  remained  alive,  though  not  for  a  long 
period,  if  the  soil  were  dried  and  reduced  to  dust.  If,  however,  the 
bacillus  is  added  to  a  well-moistened  but  not  sloppy  cultivated  soil, 
it  rapidly  dies,  and  is  usually  not  obtainable  two  days  after  being 
sown  in  it,  and  its  disappearance  appears  to  be  more  rapid  the  higher 
the  temperature,  which  is  probably  due  to  the  rapid  growth  of 
ordinary  soil  bacteria.  If  the  cultivated  soil  is  not  made  very  moist 
when  the  B.  typhosus  is  added,  the  organism  can  be  recovered  from 
the  soil  up  to  twelve  days  after  it  has  been  added.  Lastly,  if  this 
bacillus  is  added  to  natural  uncultivated  soils  which  have  not  been 
sterilised,  it  ceases  to  exist  within  twenty-four  hours.  Martin  holds 
that  the  reason  of  the  rapid  disappearance  of  the  typhoid  bacillus 
from  natural  unsterilised  soils  is  probably  twofold.  First,  there  is 
the  antagonism  of  the  soil  bacteria,  many  of  which  are  putrefactive ; 
and  secondly,  the  typhoid  bacillus  requires  for  its  growth  nitrogenous 
substances,  usually  in  the  form  of  proteids.  Cultivated  soil  is  dis- 
tinguished from  uncultivated  soil  by  containing  more  nitrogenous 
organic  matter  in  the  form  of  nitrates  and  ammonia,  and  also  more 
partially  changed  proteid  substances.  Hence  it  is  a  more  favourable 
environment  for  the  typhoid  bacillus.  As  a  general  result  of  these 
investigations,  it  may  be  concluded  that  the  typhoid  bacillus  has, 
commonly,  only  a  short  existence  in  the  soil,  being  destroyed  by  the 
products  of  the  putrefactive  bacteria  which  exist  in  most  cultivated 
soils.f 

*  Brit.  Med.  Jour.,  1898,  i.,  pp.  69-71. 

t  Reports  of  Medical  Officer  to  the  Local  Government  Board,  1898-99,  pp.  382412  ; 
1899-1900,  pp.  525-548  ;  1900-01,  pp.  487-510. 


148  BACTERIA  IN  THE  SOIL 

Lastly,  we  have  the  results  of  the  investigations  of  Firth  and 
Horrocks,  who  conclude  that  the  typhoid  bacillus  is  able  to  assume  a 
vegetative  existence  in  ordinary  soils  and  in  sewage-polluted  soils  for 
as  long  as  seventy-four  days.  They  further  maintain  that  the 
controlling  factor  is  an  excess  or  deficiency  of  moisture  in  the  soil 
rather  than  organic  nutritive  material.  From  dry  fine  sand  the 
bacillus  was  recovered  after  twenty-five  days ;  from  moist  fine  sand, 
after  twelve  days ;  from  damp  (rain-water)  ordinary  soil,  after  sixty- 
seven  days ;  from  damp  (sewage)  ordinary  soil,  after  fifty-three  days ; 
and  from  ordinary  soil  dried  to  the  state  of  dust,  after  twenty-four 
days.  In  peat  the  bacillus  lives  apparently  only  a  few  days.*  Firth 
and  Horrocks,  therefore,  arrive  at  a  different  conclusion  from  Martin, 
namely,  that  the  typhoid  bacillus  is  able  to  assume  a  vegetative  or 
saprophytic  existence  for  considerable  periods  outside  the  body;  that 
it  can  survive  ordinary  earth  for  over  two  months,  whether  the  soil  be 
virgin  or  polluted  with  sewage,  or  frozen  hard ;  and  that,  therefore,  it 
follows  that  outbreaks  of  enteric  fever  may  be  due  to  the  dissemina- 
tion (for  example  by  wind  or  flies)  of  infective  soil  dust.  Pfuhl  of 
Berlin  has  arrived  at  results  confirmatory  of  these  experiments. 
From  moist  garden  earth  he  recovered  the  typhoid  bacillus  eighty-eight 
days  after  inoculation,  from  dry  sand  after  twenty-eight  days,  and 
from  moist  peat  twenty-one  days.")* 

On  the  whole  it  would  appear  that  whilst  much  valuable  research 
has  been  accomplished,  it  cannot  be  said  that  the  relation  of  the 
typhoid  bacillus  to  soil  is  understood.  Some  further  light  on  the 
subject  is  obtained  from  researches  carried  out  in  relation  to  the 
bacterial  condition  of  sewage-treated  land  and  made-up  refuse  soil, 
and  brief  reference  may  be  made  to  such  work. 

Various  workers  have  carried  out  experiments  with  the  object  of 
ascertaining  whether  in  the  surface  layers  of  soil,  after  it  has  had 
sewage  upon  it,  certain  microbes  characteristic  of  sewage  retain  their 
vitality  for  any  considerable  length  of  time ;  what,  in  short,  was  the 
fate  of  such  organisms  as  B.  coli,  B.  enteritidis  sporogenes,  etc.,  when 
sown  broadcast  on  soil.  For  if  their  fate  be  known,  not  only  would 
light  be  thrown  upon  questions  of  sewage  treatment,  and  the  pollu- 
tion of  soil  which  might  in  turn  affect  water  supplies,  but  indication 
would  be  obtained  as  to  the  possibility  of  disease  germs  maintaining 
their  existence  in  soil,  and  eventually  infecting  man.  Chief  among 
such  investigations  in  England  have  been  those  of  Dr  A.  C.  Houston,! 
whose  conclusions  are  briefly  as  follows : — 

(1)  The  addition  of  sewage  to  an  ordinary  garden  soil  does  not 

*  Brit.  Med.  Journ.,  1902,  ii.,  pp.  936-943. 
t  Zeti.  f.  Hyg.,  1902,  Bd.  xl.,  Heft  3,  p  555. 

$  Report  of  Medical  Officer  to  Local  Government  Board,  1900-01,  App.  No.  4, 
p.  405 ;  1901-02,  App.  No.  6,  p.  455. 


POLLUTED  SOIL  149 

seemingly  lead  to  other  than  a  temporary  increase  of  the  sewage 
microbes  at  the  expense  of  the  soil  microbes,  the  ordinary  soil 
bacteria  ousting  the  sewage  microbes  in  the  struggle  for  existence. 
But  the  addition  of  sewage  to  a  sandy  soil  leads  to  an  enormous 
increase  in  the  total  number  of  microbes  as  compared  with  the 
number  originally  present  in  the  soil,  which  does  not  revert  to  its 
original  state  for  some  months. 

(2)  The   addition  of  sewage  to  garden   soil   tends  primarily   to 
increase  the  ratio  of  total  number  of  bacteria  to  spores  of  aerobic 
bacteria,  though  the  alteration  is  apt  to  be  soon  lost. 

(3)  The  addition  of  sewage  to  a  soil  leads  to  an  increase  for  a  time 
in   the  number   of    certain   kinds   of   bacteria,  namely:   (a)   indol- 
producing  bacteria ;  (&)  gas-producing  organisms ;  (e)  the  spores  of 
B.  enteritidis  sporogenes  ;  (d)  B.  coli  communis  and  its  allies ;  and  (e) 
streptococci.     The  occurrence  of  true  streptococci  in  soil  indicates,  in 
Houston's  opinion,  extremely  recent  contamination.     Whatever  inter- 
pretation be  placed  on  these  facts,  it  is  evident  that  they  indicate  that 
pathogenic  organisms  such  as  the  typhoid  bacillus  do  not  maintain 
their  vitality  in  the   surface  layers  of   soil  for  more   than  a  brief 
period.      Further,  it   is  evident   that   some   kinds   of   soils   heavily 
polluted  with  excremental  matter  tend  to  purify  themselves,  so  far  as 
non-sporing  bacilli  of  intestinal  origin  are  concerned. 

On  the  bacterial  content  of  made-soil  Dr  Savage  of  Colchester  has 
carried  out  some  work.  The  samples  of  such  soil  were  collected  with 
a  sterilised  Frankel's  borer,  and  the  samples  transmitted  to  the 
laboratory  in  sterile  Petri  dishes.  Each  sample  was  then  thoroughly 
broken  up  and  uniformly  mixed  in  the  Petri  dishes  by  means  of 
sterile  spatulas.  Ten  grammes  were  weighed  on  sterile  glazed  paper, 
and  added  to  100  c.c.  of  sterile  water  in  a  large  flask,  and  thoroughly 
mixed.  The  contents  of  the  flask  were  allowed  to  settle  for  five 
minutes,  and  without  disturbing  the  sediment  1  c.c.  of  the  water  was 
taken  up  and  added  to  further  quantities  of  sterile  water  for  dilution 
purposes.  The  examination  was  then  carried  out  in  the  ordinary  way, 
with  a  view  of  determining  (a)  total  number  of  organisms  present ; 
(b)  number  of  B.  coli  ;  (c)  number  of  B.  mycoides,  and  of  Bismark- 
brown  Cladothrix;  and  (d)  the  smallest  quantity  of  soil  producing 
indol  in  one  week  at  37°  C.  grown  in  peptone  water  solution. 

As  a  result  of  these  experiments,  Dr  Savage  reports  that  at  a 
depth  of  two  feet  in  mounds  of  tip-refuse  deposited  on  damp  imper- 
vious clay,  putrefaction  and  concurrent  purification  takes  place  fairly 
rapidly  for  the  first  two  or  three  years.  The  organisms  present  in 
the  refuse  as  deposited  rapidly  decrease  at  the  same  time,  but  after 
two  to  three  years  increase,  apparently  due  to  the  invasion  of  ordinary 
soil  organisms.  After  two  to  three  years,  purification  at  this  depth 
takes  place  extremely  slowly,  and  samples  nine  to  ten  years  old  give 


150  BACTERIA  IN  THE  SOIL 

results  very  little  different  from  four  to  five  year  old  samples.  The 
B.  coli  readily  dies  out  in  such  refuse  heaps,  from  which  Dr  Savage 
infers  that  the  B.  typhosus,  being  a  less  resistant  organism,  would  still 
more  rapidly  die  out,  and  that  therefore  "the  danger  of  specific 
typhoid  contamination  from  building  on  such  made-soil  can  be 
neglected."  * 

From  what  has  been  said,  it  will  be  seen  that  though  a  consider- 
able amount  of  knowledge  has  been  obtained  respecting  bacteria  in 
the  soil,  it  may  be  conjectured  that  actually  there  is  still  a  great  deal 
to  ascertain  before  the  micro-biology  of  soil  is  in  any  measure  com- 
plete or  even  intelligible.  The  mere  mention  of  the  bacilli  of  tetanus 
and  typhoid  in  the  soil,  and  their  habits,  nutriment,  and  products 
therein,  not  to  mention  the  work  of  the  economic  bacteria,  is  to  open 
up  to  the  scientific  mind  a  vast  realm  of  possibility.  It  is  scarcely  too 
much  to  say  that  a  fuller  knowledge  of  the  part  which  soil  plays  in 
the  culture  and  propagation  of  bacteria  may  suffice  to  modify 
many  views  in  preventive  medicine.  True,  our  knowledge  at  the 
moment  is  rather  a  heterogeneous  collection  of  isolated  facts  and 
theories,  some  of  which,  at  all  events,  require  ample  confirmation ; 
yet  still  there  is  a  basis  for  the  future  which  promises  much  con- 
structive work. 

*  Jour,  of  Sanitary  Institute,  1903,  vol.  xxiv.,  pt.  iii.,  pp.  442-458. 


CHAPTEE  VI 


THE  BACTERIOLOGY  OF  SEWAGE  AND  THE  BACTERIAL 
TREATMENT  OF  SEWAGE 

Composition  of  Sewage — Quantity  and  Quality  of  Bacteria  in  Sewage— Treatment 
of  Sewage:  (1)  Disposal  without  Purification;  (2)  Chemical  Treatment; 
(3)  Bacterial  Treatment  —  Evolution  of  Bacterial  Methods  —  Septic  Tank 
Method— Contact  Bed  Method— Manchester  Experiments— Effect  of  Bacterial 
Treatment  on  Pathogenic  Organisms. 

THE  relation  of  bacteria  to  sewage  has  during  the  last  twenty-five 
years  assumed  a  position  of  the  first  importance.  This  is  due, 
generally  speaking,  to  three  causes.  In  the  first  place,  our  knowledge 
of  the  economic  function  of  bacteria  present  in  sewage  has  increased 
in  a  very  large  measure  in  recent  years.  Secondly,  as  the  population 
has  tended  to  gravitate  to  cities,  the  problem  of  a  pure  water  supply, 
free  from  sewage  pollution,  has  become  infinitely  more  complicated 
than  was  the  case  in  rural  communities  in  the  past.  How  often 
sewage,  from  sewage  or  cesspools,  gains  access  by  means  of  direct 
connection  or  percolation  to  drinking  water,  the  history  of  typhoid 
epidemics  and  similar  outbreaks  in  this  country  only  too  fully  records. 
And  thirdly,  practical  issues  have  now  arisen  in  connection  with  the 
bacterial  treatment  of  sewage.  In  order  to  understand  the  bacteri- 
ology of  sewage  and  its  practical  lessons,  we  may  first  briefly  consider 
the  quality  and  constitution  of  sewage  as  regards  its  bacterial  content, 
and  then  proceed  to  discuss  its  biological  treatment. 

The  Constitution  of  Sewage 

It  is  impossible  to  lay  down  any  exact  standard  of  the  chemical 
and  bacterial  quality  of  sewage.  The  quality  will  differ  according  to 
the  size  of  the  community,  the  inclusion  or  otherwise  of  trade-effluents 
and  waste  products,  the  addition  of  rain  and  storm  water,  and  other 

151 


152  BACTERIAL  TREATMENT  OF  SEWAGE 

similar  physical  conditions.*  Moreover,  the  sewage  itself  is  con- 
stantly undergoing  rapid  changes  owing  to  fermentation,  and  the 
competition  of  micro-organisms  and  the  effect  of  their  products.  It 
is  clear  that  they  are  the  chief  agents  in  setting  up  fermentative  and 
putrefactive  changes,  for  if  sewage  be  placed  in  hermetically  sealed 
flasks  and  sterilised  by  heat  it  will  be  found  that  these  changes  do 
not  occur.  Hence  it  will  be  at  once  apparent  that  no  exact  or  hard- 
and-fast  formula  can  be  laid  down.  Eespecting  the  chemical  con- 
dition, with  which  we  have  but  little  to  do  here,  we  may  shortly  say 
that  the  chief  characteristic  of  sewage  is  its  enormous  amount  of 
contained  organic  matter  (yielding  saline  and  albuminoid  ammonia, 
etc.)  in  suspension  or  in  solution.  But  there  are  in  addition  various 
inorganic  substances,  and  hence  it  is  customary  to  subdivide  the 
chemical  constituents  into  (a)  organic  matter  in  suspension  ;  excreta, 
etc. ;  (b)  organic  matter  in  solution ;  (c)  inorganic  matter  in  suspen- 
sion, such  as  sand,  grit,  street  and  road  washings,  gravel,  etc. ;  and 
(d)  inorganic  matter  in  solution,  which  is  not  great  in  amount, 
but  includes  phosphates,  one  of  the  favouring  agencies  of  sewage 
fungus.  "We  may  summarily  classify  the  constituents  of  sewage  as 
follows : — 

(a)  IZxcretory  substances,   composed  of   solid  excreta  and  urine. 
The  former  consist  of  nitrogenous  partly-digested  matter,  together 
with  vegetable  non-nitrogenous  residues  of  food.      The  former  are 
easily  broken .  down ;    but   the  latter  are   only  gradually  attacked 
(chiefly  by  the  anaerobic  bacteria),  and  broken  down  into  soluble 
compounds   foetidly  odorous,  and  into   small   black   masses,   which 
slowly  deposit  as  black  sludge. 

(b)  Household  waste,  solid  substances,  washings,  etc. 

(c)  Rain  and  storm  water  of  varying  amount,  according  to  season. 

(d)  Grit,  gravel,  sand,  etc. 

(e)  Manufacturing  waste  products  in  certain  localities. 

Turning  to  the  bacterial  content,  it  will  at  once  occur  to  us  that 
such  a  large  quantity  of  organic  matter  as  sewage  contains,  and  in 
which  decomposition  is  constantly  taking  place,  will  afford  an  almost 
ideal  nidus  for  micro-organic  life.  There  is,  indeed,  but  one  reason 
why  such  a  medium  is  not  absolutely  ideal  from  the  microbe's  point 
of  view,  and  that  reason  is  that  in  sewage  the  vast  numbers  of 
bacteria  present  make  the  struggle  for  existence  exceptionally  keen. 
The  source  of  the  organisms  is  most  largely  the  organic  dejecta  chiefly 
constituting  the  sewage,  but  there  are  in  addition  the  organisms  of 
the  air  and  extraneous  fluids  and  substances  found  in  sewage.  The 
result  of  Jordan's  f  investigations  into  sewage  gave  an  average  of 
708,000  living  bacteria  per  c.c.,  his  highest  result  being  3,963,000 

*  Analyst,  199,  xxiii.,  1898. 

f  Report  of  State  Board  of  Health,  Massachusetts,  1890. 


BACTERIA  IN  SEWAGE  153 

per  c.c.  He  obtained  higher  figures  during  the  summer  months 
than  at  other  times ;  but  in  any  case  his  average  was  extremely  low. 
Laws  and  Andrewes  *  found  that  London  crude  sewage  varied 
from  2,781,650  to  11,216,666  micro-organisms  per  c.c.  "It  will 
thus  be  seen,"  they  conclude,  "  that  very  wide  variations  exist  in  the 
total  number  of  micro-organisms  present  in  sewage  at  different  times 
and  in  different  places.  Temperature  is  one  important  factor  in 
determining  the  rapidity  of  their  reproduction,  and  hence  their 
increase  in  numbers;  dilution  of  the  sewage  by  rainfall  must  also 
exert  a  marked  influence."  Houston  •(•  has  also  examined  the  sewage 
of  London,  and  found,  in  1898,  that  the  Barking  crude  sewage  con- 
tained an  average  of  nearly  four  millions  of  organisms  per  c.c.  and 
the  Crossness  crude  sewage  three  and  a  half  millions  per  c.c.  In 
1899  the  same  observer  J  reported  7,357,692  bacteria  per  c.c.  as  the 
average  in  the  sewage  at  the  Crossness  outfall.  On  one  occasion  he 
records  19,500,000  micro-organisms  as  present  in  one  cubic  centi- 
metre. In  1900,  Houston  reported  similar  figures,  and  on  occasion 
as  many  as  1,900,000  B.  coli  per  c.c.  in  crude  sewage.  He  further 
added  some  records  as  to  the  number  of  bacteria  from  crude  sewage 
growing  at  blood-heat  and  room  temperature.  In  the  former 
case  he  found  as  many  as  6,830,000,  and  in  the  latter  11,170,000 
per  c.c.§ 

Not  only  are  the  numbers  incredibly  large,  but  we  also  find  an 
extensive  representation  of  species,  including  both  saprophytes  and 
parasites,  non-pathogenic  and  pathogenic.  Many  of  these  are  known 
as  "liquefying"  bacteria  (from  the  power  which  they  possess  of 
liquefying  or  peptonising  nutrient  gelatine  used  as  a  culture  medium), 
and  this  is  one  of  the  features  of  putrefactive  bacteria.  Bacilli  pre- 
ponderate over  micrococci  in  actual  numbers,  and  in  numbers  of 
species  present.  There  are  also  many  spores.  Dr  Houston  has 
tabulated  these  results  in  his  Third  Keport  (1900)  from  which  it 
appears  that  there  are  about  340  spores  per  c.c.,  and  1,076,923  lique- 
fying bacteria  per  c.c.  Moulds  are  but  rarely  found  in  sewage, 
though  common  in  sewer  air. 

It  is  probable  that  the  investigations  made  into  the  contained 
bacteria  of  sewage  have  up  to  the  present,  excellent  though  they 
have  been,  only  revealed  those  species  of  bacteria  which  occur  in 
considerable  abundance.  So  though  it  is  impossible  to  make  any 
very  complete  record  as  regards  the  species  of  bacteria  present  in 

*  Report  on  the  Result  of  Investigations  on  the  Micro-organisms  of  Sewage,  London 
County  Council,  1894. 

t  "  Filtration  of  Sewage,"  Report  on  the  Bacteriological  Examination  of  London 
Crude  Sewage  (First  Report),  London  County  Council,  1898. 

J  "  Bacterial  Treatment  of  Crude  Sewage "  (Second  Report),  London  County 
Council,  1899. 

§  Ibid.  (Third  Report),  1900,  p.  59. 


154 


BACTERIAL  TREATMENT  OF  SEWAGE 


sewage,  we  may  attempt  a  provisional  list  of  normal  types  of  sewage 
bacteria  *  as  follows : — 

1.  Bacillus  coli  communis  and  all  its  varieties  and  allies.     Houston 
reports  as  many  as  600,000  B.  coli  per  c.c.  in  London  sewage. 

2.  The  Proteus  family — Proteus  vulgaris,  P.  Zenkeri,  P.  mirabilis, 
and  P.  cloacinus,  first  isolated  from  putrid  meat  by  Hauser,  isolated 
from  sewage  by  Jordan,  etc.     Houston  also  reports  that  frequently 
there  may  be  100,000  "sewage  proteus"  present  in  one  c.c.     This  is 
an  aerobic,  non-chromogenic,  actively  motile,  and  rapidly  liquefying 
bacillus  with  round  ends,  one  flagellum,  and  no  spore  formation.     It 
differs  in  essential  particulars  from  the  P.  vulgaris.     Some  of  the 
cultures  were  pathogenic  to  guinea-pigs  (Plate  14). 

3.  Bacillus  enteritidis  sporogenes  of  Klein.     The  number  of  spores 
of  this  organism  found  in  London  sewage  by  Houston  varied  from 
10  to  1000  per  c.c.,  thus  often  exceeding  in  number  the  total  number 
of  spores  of  aerobic  bacilli.     The  relative  numbers  of  B.  coli  and  the 
spores  of  B.  enteritidis  sporogenes  in  crude  sewage  have  been  demon- 
strated by  Klein  and  Houston  in  the  following  table : — 


No.  of  Spores 

Sample  of  Crude  Sewage. 

No.  of  Bacteria 
per  c.c. 

No.  of  B.  coli 
per  c.c. 

of 

B.  ent.  sporog. 

per  c.c. 

1.  Chiefly  domestic  sewage           .        . 

14,240,000 

260,000 

2000 

2.  Mixed  sewage           .         .       •  . 

7,800,000 

180,000 

200 

3.  Chiefly  domestic  sewage  . 
4.  Mixed  sewage  and  trade-effluent 

4,800,000 
36,000,000 

500,000 
1,100,000 

2000 
.400 

5.  Hospital  sewage       .... 

2,800,000 

200,000 

30 

6.  Domestic  sewage  and  trade-effluent 

4,100,000 

500,000 

56 

7.  Domestic  sewage      .        .       ..:    '  .: 

28,100,000 

2,000,000 

50 

8.  Mixed  sewage  .         .        . 

21,100,000 

1,000,000 

35 

*  The  methods  adopted  for  making  a  quantitative  and  qualitative  examination 
of  sewage  are  precisely  analogous  to  those  used  in  milk  research.  Dilution  with 
sterilised  water  previous  to  plating  out  on  gelatine  in  Petri  dishes  is  essential  (1  c.  c. 
to  10,000  c.c.  of  sterile  water,  or  some  equally  considerable  dilution),  otherwise  the 
large  number  of  germs  would  rapidly  liquefy  and  destroy  the  film.  The  plate 
should  be  incubated  at  a  definite  temperature,  which  is  usually  20°  C.  Special 
methods  must  be  used  for  the  isolation  of  special  organisms,  phenol-gelatine  ( '1  c.c. 
of  a  5  per  cent,  solution  of  phenol  to  every  10  c.c.  of  gelatine).  Eisner  medium, 
Parietti  broth,  indol-reaction,  and  "shake"  cultures  in  gelatine  (for  testing  gas- 
production)  must  often  be  restored  to  for  certain  species.  Spores  in  sewage  may 
be  isolated  by  adding  1  c.c.  of  diluted  sewage  (1-10)  to  10  c.c.  of  melted  gelatine  in 
a  test-tube,  and  heating  the  mixture  to  80°  C.  for  ten  minutes  before  pouring  out 
into  the  Petri  dish.  This  temperature  kills  all  the  bacilli,  but  leaves  the  spores 
untouched.  The  same  plan  is  adopted  in  principle  for  separating  B.  enteritidis 
sporogenes :  a  small  quantity  of  sewage  is  added  to  15  c.c.  of  fresh  sterile  milk,  which 
is  heated  at  80°  C.  for  ten  minutes,  and  then  incubated  at  37°  C.  anaerobic-ally  in  a 
Buchner  tube.  J3.  coli  communis  is  grown  in  phenol-broth  for  twenty-four  hours, 
and  then  plated  out  on  phenol-gelatine. 


PLATE  14. 


SEWAGE  PROTEUS  (Houston). 

Film  preparation  from  agar  culture,  24  hours  at  20°  C. 
X  1000. 


SEWAGE  PROTEUS  (Houston). 
Gelatine  plate  culture,  48  hours'  growth  at  20°  C.    (Natural  size). 


[To  face  page  154. 


SEWAGE  BACTERIA  155 

4.  Sewage  Streptococci. — Laws  and  Andrewes,  Houston,  Horrocks, 
and   others   have  isolated   streptococci  from   crude   sewage,   which 
appear  to  be  normal  sewage  organisms,  and  as  such  may  be  taken, 
when  present  in  water,  to  indicate  contamination,  and,  if  accompanied 
by  B.  coli,  recent  and  dangerous  contamination.     Staphylococci  have 
also  been  frequently  isolated.     Houston  has  described  some  twenty 
streptococci   as   present  in   London   sewage.      They  are   generally 
present  in  crude  sewage  in  numbers  not  less  than   1000  per  c.c. 
These  sewage  streptococci  are  delicate,  and  readily  lose  their  vitality 
and  die.     They  are  probably  little  prone  to  enter  on  a  saprophytic 
phase  or  to  multiply  to  any  great  extent,  if  at  all,  under  such  condi- 
tions as  prevail  in  sewage.     They  are  present  in  the  intestinal  dis- 
charge of  animals,  and  comprise  highly  pathogenic  organisms.     They 
are   usually  absent  in   pure   waters   and   virgin  soils,   and   waters 
recently  polluted   with   excremental  matters.     They  stain   well  by 
Gram's  method.     The  majority  form  short  chains,  which  sometimes 
cohere  in  masses.      They  grow  well  at  blood-heat  in  the  ordinary 
media,  producing  acid  in  milk  without  clotting  it.*     Some  streptococci 
from  sewage  coagulate  milk  (Plate  15). 

5.  Liquefying    bacteria,   e.g.   Bacillus    superficialis   (Jordan),   B. 
frondosus  (Houston),  B.  Jiyalinus  (Jordan),  B.  delicatulus  (Jordan), 
B.  cloacce  (Jordan),  B.  fluorescens  stercoralis,  B.  menibraneus  patulus, 
B.  capillareus  (Houston),  B.  cloacce  fluorescens  (Laws  and  Andrewes), 
various  forms  of  Clostridium  and  the  typical  B.  mesentericus  (Plate  16). 

6.  Non-liquefying  bacteria,  e.g.  Bacillus  subtilissimus,  B.fusiformis 
(Houston),  B.  rubescens  (Jordan),  B.  pyogenes  cloacinus  (Klein  •(*). 

We  have  not  included  in  the  above  classification  any  bacteria 
virulently  pathogenic  to  man.j  Doubtless,  such  species  (e.g.  Bacillus 
typhosus)  not  infrequently  find  their  way  into  sewage.  But  they  are 
not  for  various  reasons  normal  habitants,  and  though  they  struggle 
for  survival,  the  keenness  of  the  competition  among  the  dense  crowds 
of  saprophytes  makes  existence  for  a  continuous  period  in  sewage 
almost  impossible  for  them.  In  the  investigation  to  which  reference 
has  already  been  made,  Laws  and  Andrewes  devoted  some  attention 
to  the  behaviour  of  B.  typhosus  in  sewage.  They  found  that  this 
bacillus  was  unable  to  grow,  indeed  quickly  perished,  in  sewage 
sterilised  by  filtration  and  heat,  whereas  the  B.  coli  is  able  to  increase 
and  multiply  in  such  a  medium.  Sewage,  therefore,  even  in  the 
absence  of  the  normal  micro-organisms  which  it  contains,  is  an 

*  Bacterial  Treatment  of  Crude  Sewage— Third  Report  to  the  London  County 
Council,  by  Dr  Houston,  1900,  pp.  60-68  ;  Royal  Commission  on  Sewage  Disposal, 
Second  Report,  1902,  p.  25. 

t  See  British  MedicalJournal,  1899,  vol.  ii.,  p.  69. 

J  Bacillus  enteritidis  sporogenes,  B.  pyogenes  cloacinus,  and  other  organisms  have 
been  held  responsible  for  diarrhoea,  abscess  formation,  etc.,  but  they  cannot  yet 
be  compared  with\B.  typhosus  as  regards  pathogenic  effect. 


156  BACTERIAL  TREATMENT  OF  SEWAGE 

unfavourable  medium  for  the  growth  of  the  typhoid  bacillus,  which 
in  all  probability  would  die  out  in  a  few  days'  time.  In  crude 
unsterilised  sewage  it  is  clear  that  owing  to  competition  and  the 
inimical  effect  some  of  the  non-pathogenic  species  have  upon  B. 
typhosus*  that  the  death  of  that  organism  is,  in  sewage,  "  probably 
only  a  matter  of  a  few  days  or  at  most  one  or  two  weeks."  MacConkey 
found  that  in  sterilised  crude  sewage  inoculated  with  the  B.  typhosus, 
this  bacillus  is  recoverable  in  seventeen  days,  though  it  does  not  appear 
to  multiply.  In  ordinary  crude  sewage  so  inoculated,  the  bacillus 
was  recoverable  after  thirteen  days.f 

Of  the  organisms  which  we  have  named  as  normally  present  in 
sewage,  it  is  unnecessary  to  speak  in  detail,  with  the  exception  of  the 
Bacillus  enteritidis  sporogenes  of  Klein.!  This  bacillus  is  credited 
with  being  a  causal  agent  in  diarrhoea,  and  has  been  isolated  by 
Dr  Klein  from  the  intestinal  contents  of  children  suffering  from 
autumnal  diarrhoea,  and  from  adults  having  "  English  cholera."  It 
has  readily  been  detected  in  sewage  from  various  localities,  and  also 
in  some  sewage  effluents.  It  has  been  separated  from  ordinary 
milk,  even  from  what  was  termed  by  the  trade  "  sterilised "  milk. 
The  biological  characters  of  this  bacillus  are  briefly  as  follows.  It 
is  in  thickness  about  equal  to  the  bacillus  of  quarter-evil,  thicker  and 
shorter  than  the  bacillus  of  malignant  oedema,  and  standing  therefore 
between  the  latter  and  the  bacillus  of  anthrax.  It  is  motile  and 
possesses  flagella,  but  does  not  assume  a  thread  form.  It  readily 
forms  spores,  which  develop,  as  a  rule,  near  the  ends  of  the  rods, 
and  are  thicker  than  the  bacilli.  They  can  withstand  a  tempera- 
ture of  80°  C.  for  fifteen  minutes.  The  bacillus  takes  the  Gram 
stain.  In  various  media  it  produces  gas  rapidly.  Particularly 
is  this  so  in  milk.  It  is  an  anaerobe,  and  may  be  isolated  by  the 
following  method.  A  small  quantity  of  the  suspected  matter  is 
added  to  a  tube  of  fresh  sterilised  milk,  which  is  then  heated  in  a 
water-bath  to  80°  C.  for  fifteen  minutes.  It  is  then  cooled  and 
incubated  at  blood-heat  in  a  Buchner's  tube  (see  p.  478).  In  twenty- 
four  hours  the  milk  is  coagulated  into  white  stringy  masses  and  small 
casein  coagula,  whilst  a  large  portion  of  the  test-tube  is  filled  with 
gas  or  a  thick,  watery,  slightly-turbid  whey.  It  is  necessary  to 
differentiate  the  B.  enteritidis  sporogenes  from  the  bacilli  of  malignant 
oedema  and  symptomatic  anthrax  and  the  Bacillus  lutyricus  of  Botkin. 
For  such  differentiation  it  is  important  to  remember  that  the 
enteritidis  organism  (a)  stains  by  Gram's  method,  (&)  in  gelatine  culture 

*  Klein  reports  that  although  B.  typhosus  can  live  in  crude  sewage,  it  is  only 
for  a  short  period.  When  sewage  is  diluted  with  large  quantities  of  water  the  case 
is  different. 

f  Royal  Commission  on  Sewage  Disposal,  Second  Report,  1902,  p.  62. 

£  Annual  Report  of  the  Medical  Officer  of  the  Local  Government  Board,  1897-98, 
pp.  210-250. 


PLATE  15. 


, 


r 


SEWAGE  STREPTOCOCCUS,  from  Effluent.  (Houston.) 

j     From  broth  culture,  48  hours  at  38°  C.    Stained  by 
Gram's  method,     x   1000. 


Streptococcus  pyogenes. 

Film  culture  from  broth  culture.    Stained  by  Gram 
method,     x  1000. 


SEWAGE  STREPTOCOCCUS,  from  Crude  Sewage.     (Houston.) 

From  broth  culture,  48  hours  at  38°  C.    Stained  by  Gram's  method, 
x  1000. 


[To  face  page  156. 


SEWAGE  BACTERIA  157 

shows  no  lateral  offshoots,  and  (c)  possesses  different  pathological 
characters  on  inoculation.  If  1  c.c.  of  whey  from  a  milk  culture  be 
inoculated  into  a  guinea-pig  (200-300  grammes)  a  swelling  appears 
in  the  groin  after  six  hours,  which  extends  to  the  abdomen  and  thigh. 
The  animal  is  usually  dead  in  eighteen  to  twenty-four  hours  with 
gangrene  of  the  subcutaneous  tissue  and  offensive  sanguineous  exuda- 
tion. These  characteristics,  coupled  with  the  morphological  and  bio- 
logical features,  are  sufficient  for  differentiation  purposes  (see  also 
p.  307). 

Houston  has  shown  that  the  cholera  bacillus,  B.  pyocancus,  and 
Staphylococcus  pyogenes  aureus  are  capable  of  retaining  their  vitality 
in  crude  sewage  in  competition  with  the  very  numerous  bacteria 
normally  present.*  The  bacillus  of  anthrax,  and  still  more  so  its 
spores,  can  also  live  in  sewage  and  sewage  effluents  (Houston).f 

Whilst  we  cannot  here  enter  more  fully  into  an  account  of  the 
bacteria  found  in  sewage  or  their  functions,  it  is  necessary  to  remark 
upon  one  important  feature.  A  large  number  of  these  organisms 
which  we  have  named  as  normal  inhabitants  of  sewage  fulfil  as  their 
main  function  the  process  of  decomposition  and  denitrification,  that 
is  to  say,  their  role  is  to  break  down,  by  means  of  putrefaction,  the 
organic  compounds  constituting  sewage.  For  example,  urea  which 
is  abundantly  present  in  sewage  is  thus  transformed  with  extra- 
ordinary rapidity  by  several  different  forms  of  bacteria. 

By  way  of  summary  we  may  quote  Houston's  account  of  the 
"  standard  "  of  crude  sewage.  Crude  sewage  usually  contains,  at  least, 
in  one  cubic  centimetre — 

(a)  1-10  million  bacteria. 

(b)  100,000  B.  coli  or  closely  allied  forms. 

(c)  100  spores  of  B.  enteritidis  sporogenes. 

(d)  1000  streptococci. 

00  Tirinr  c-c-  ig  usually  sufficient  to  produce  "gas"  in  gelatine 
shake  cultures  in  twenty-four  hours  at  20°  C. 

(/)  The  inoculation  of  animals  with  crude  sewage  always  leads 
to  a  local  reaction,  and  not  uncommonly  results  in  death.;]; 

As  we  have  already  said,  when  dealing  with  the  Bacteria  of  the 
Soil,  Nature  is  dependent  upon  the  services  of  the  "economic" 
organisms.  Dead  organic  matter  is  broken  down  as  the  result  of 
the  vital  activity  of  putrefactive  bacteria  (decomposing  and  denitri- 
fying). The  ammonia  which  is  thus  liberated  becomes  oxidised  first 
to  nitrous  and  then  to  nitric  acid  by  the  agency  of  the  nitrifying 
bacteria,  and  the  acids  by  their  action  upon  bases,  always  present, 
produce  nitrites  and  then  nitrates.  It  is  upon  these  substances  that 

*  Bact.  Treatment  of  Crude  Sewage,  Third  Report,  1900,  p.  75. 

f  Royal  Commission  on  Seioage  Disposal,  Second  Report,  1902,  p.  31. 

I  ./Wd.,1902,p.  126. 


158  BACTERIAL  TREATMENT  OF  SEWAGE 

plant  life  finds  nutriment.  That  the  carbon  is  converted  into  CO.,, 
the  hydrogen  into  water  (H20),  and  the  "  lost "  nitrogen  refixed  in 
the  soil,  we  have  already  seen. 

Now  just  as  soil  contains  these  Economic  Organisms,  whose  role 
is  to  complete  the  cycle  of  nature,  removing  the  dead  remains  of 
plants  and  animals,  and  assimilating  them  in  such  a  way  as  to  add 
to  the  fertility  of  the  soil  and  recommence  the  cycle  of  life,  so  also 
in  sewage  we  have  all  the  required  organisms  normally  present,  whose 
business  it  is  to  render  soluble  the  solid  matters,  and  to  split  up  the 
organic  compounds  into  their  simple  elements,  and  then  as  a  final 
stage  in  the  process  to  oxidise  these  elements  and  so  produce  an 
effluent  free  from  putrescible  matter,  but  containing  nitrates  and 
other  mineral  substances.*  For  practical  purposes  these  two  main 
groups  of  bacteria,  the  breakers-down  and  the  builders-up,  are  looked 
upon  as  anaerobic  or  aerobic.  The  former  are  active  in  the  absence 
of  air,  and  their  activity  effects  a  decomposition  of  complex  organic 
matter  and  allied  substances.  The  aerobes  are  most  active  in  the 
presence  of  oxygen,  and  part  of  their  business  is  to  convert  urea  into 
ammonia  and  ammonia  into  nitrate. 

From  this  brief  recital  of  the  functions  of  many  of  the  sewage 
bacteria  we  learn  that  they  have  important  operations  to  perform, 
and  that  their  presence  in  sewage,  even  in  very  large  numbers,  is 
not  matter  for  regret,  but  far  otherwise.  We  see  also  a  remarkable 
adaptation  of  those  fermentations  discovered  by  Schloesing  and 
Miintz,  in  1878,  to  be  of  such  inestimable  economic  value  in  soil. 

We  are  now  in  a  position  to  consider  the  treatment,  especially 
the  biological  treatment,  of  sewage. 

The  Biological  Treatment  of  Sewage 

Almost  from  time  immemorial  there  has  been  adopted  one  of 
three  great  methods  of  disposal  of  sewage : — 

1.  Disposal  without  purification. 

2.  Mechanical  and  chemical  separations. 

3.  Biological  methods. 

It  may  be  convenient  to  add  here  that  the  complete  purification 
of  sewage  involves  three  processes : — First,  the  process  of  clarifica- 
tion, that  is  to  say,  the  removal  of  suspended  solid  matters ;  secondly, 
an  alteration  of  the  chemical  constitution  of  organic  putrescible 

*  The  following  have  been  considered  as  the  general  conditions  which  an  effluent 
ought  to  fulfil :  (a)  It  must  contain  practically  no  solids  in  suspension  ;  (b)  it  must 
not  contain  in  solution  a  quantity  of  organic  matter  sufficient  to  seriously  absorb  the 
oxygen  from  the  stream  water  into  which  it  is  discharged  ;  (c)  it  must  not  be  liable 
to  putrefaction  or  secondary  decomposition  ;  (d)  it  must  contain  nothing  inimical  to 
microbial  growth  and  activity,  therefore  it  must  not  be  treated  with  strong  anti- 
septics ;  (e)  it  must  not  contain  pathogenic  organisms. 


PLATE  16. 


Jjacillus  mesentericus,  Sewage  variety  (No.  i.)    (Houston.) 
Film  preparation  from  agar  culture,  20  hours  at  20°  C.      x  1000. 


Bacillus  mesentericus,  Sewage  variety  (No.  i.)    (Houston.) 
Gelatine  plate  culture,  20°  C.    (Natural  size.) 


[To  face  page  158. 


OF  THE 

UNIVERSITY 

or 


SEWAGE  DISPOSAL  159 

matter  in  solution  in  the  sewage,  so  that  such  putrescible  matter 
appears  in  the  effluent  in  a  form  which  will  not  undergo  any  further 
putrefactive  change ;  and  thirdly,  the  removal  of  disease-producing 
bacteria,  which  will  be  present  in  practically  all  crude  town  sewage. 

These  results  are  not  obtained  equally  by  the  various  methods 
employed,  but  it  will  be  best  to  consider  each  of  these  separately. 

1.  Disposal  without  Purification. 

Various  antiquated  forms  of  carrying  out  this  mode  have  been 
used.  Seaside  places  have  often  been  content  to  carry  their  un- 
treated sewage  out  to  sea.  Towns  situated  on  the  banks  of  rivers 
have  frequently  by  means  of  a  conduit  conveyed  sewage  into  the 
running  stream.  There  is  nothing  necessarily  objectionable  in  this 
mode  of  disposal,  for  both  in  the  sea  and  in  running  river  water  the 
sewage  matter  will  become  disintegrated  and  dissolved.  Yet  the 
method  is  liable  to  give  rise  to  very  serious  nuisance,  unless  the 
conditions  requisite  for  solution  are  carefully  studied.  Nuisances 
may  arise  in  respect  to  the  pollution  of  bathing  grounds,  or  actually 
injurious  effect  upon  the  health  of  the  population  on  the  banks  of 
the  river,  or  by  injury  to  fish  (by  reducing  the  oxygen  in  the  water, 
destroying  the  food  of  fish,  admitting  poisonous  matters  into  the 
water,  or  by  suspended  matters  clogging  the  gills  of  fish).  In  a 
general  way  it  may  be  said  that  before  the  admission  of  sewage  into 
any  body  of  water  is  permissible,  two  points  require  consideration, 
namely,  the  removal  as  far  as  practicable  of  the  matters  in  suspension 
in  the  sewage,  and  the  sufficiency  of  dissolved  oxygen  in  the  water 
completely  to  prevent  any  putrefaction.  Broadly,  also,  it  may 
be  said  that  for  towns  situated  on  non-tidal  streams  some  form  of 
bacterial  treatment  is  preferable.  Towns  on  tidal  rivers  require  as 
a  rule  a  chemical  precipitation  process.* 

*  Foulerton  has  recently  drawn  attention  to  a  modified  chemical  precipitation 
process  treatment  of  sewage  which  is  to  be  discharged  into  a  tidal  water,  which 
may  be  carried  out  as  follows  : — The  effluent  from  an  ordinary  chemical  precipita- 
tion process  is  distributed  continuously  over  a  coarse  "filter-bed"  by  means  of  a 
sprinkler.  In  this  way  a  thorough  aeration  of  the  effluent  before  its  discharge  into 
the  stream  is  ensured,  and  provision  is  made  for  the  complete  removal  of  all  traces 
of  solid  suspended  matter.  As  the  effluent  from  the  sedimentation  tanks  trickles 
slowly  through  the  coarse  interstices  of  the  filter-bed,  any  solid  suspended  matter 
which  has  escaped  precipitation  in  the  previous  part  of  the  process  will  be  deposited, 
and  then  dealt  with  by  bacteria.  And  in  the  result  an  effluent,  fully  oxygenated, 
free  from  the  solid  suspended  matter  of  the  crude  sewage,  and  with  the  bacteria 
originally  present  in  the  crude  sewage  considerably  decreased  in  numbers,  will  be 
discharged  into  the  stream.  The  somewhat  higher  proportion  of  dissolved  organic 
putrescible  matter  in  the  effluent  from  such  a  chemical  process,  as  compared  with 
the  proportion  which  may  obtain  in  a  good  bacterial  process,  is  probably  not  a  matter 
of  considerable  importance  in  the  case  of  tidal  waters.  —Report  on  Pollution  of  Tidal 
Fishing  Waters  by  Sewage,  1903,  p.  8. 


160  BACTERIAL  TREATMENT  OP  SEWAGE 

2.  Mechanical  and  Chemical  Separations. 

Methods  in  which  this  principle  is  applied  are  numerous ;  they 
have  generally  been  of  the  nature  of  a  "  precipitation  "  process.  Six 
to  twelve  grains  of  quicklime  have  been  added  to  each  gallon  of 
sewage,  forming  a  precipitate  of  carbonate  of  lime,  which  carries 
down  with  it  the  light,  flocculent  suspended  matter  of  the  sewage. 
The  process  is  simple  and  cheap ;  it  does  not,  however,  remove  the 
organic  matter  in  solution,  but  merely  the  solid  matters  in  suspension. 
On  the  one  hand  it  does  not  produce  a  valuable  manure;  on  the 
other  it  fails  to  purify  the  effluent.  A  score  of  other  methods  have 
been  tried  (e.g.  the  lime  and  ferrous  sulphate  treatment,  Hanson's 
process,  "  f erozone,"  amines,  electrolysis,  etc.),  but  with  the  exception 
of  electrolysis,  all  based  on  the  addition  of  chemical  substances  able 
to  precipitate  or  otherwise  change  the  organic  matter  of  the  sewage. 
All  these  methods  produce  large  quantities  of  sludge,  the  removal 
of  which,  by  pressing,  digging  into  the  land,  or  sending  out  to  sea, 
presents  many  difficulties.  But  the  chemical  processes  have  this 
advantage,  that  they  remove  disease-producing  organisms  more  per- 
fectly than  the  bacterial  process,  though  the  latter  carries  further 
the  purification  of  dissolved  organic  putrescible  matter. 

3.  Biological  Methods. 

The  biological  methods,  though  very  various,  all  have  two  common 
features.  In  the  first  place,  the  injurious  and  putrescible  substances 
in  the  sewage  are  not  merely  "  disposed  of  "  nor  yet  only  "  separated." 
They  are  destroyed.  There  is  a  destruction  of  sewage  as  sewage, 
and  a  building-up  of  new  substance  in  its  place.  Secondly,  this 
desired  effect  is  achieved,  not  by  adding  anything  to  the  sewage,  but 
~by  the  organisms  normally  present  in  the  sewage  or  in  the  medium — 
the  land  or  the  "  filtering  "  agent — upon  which  the  sewage  is  treated. 
In  short,  all  biological  processes  depend  upon  the  employment  of 
bacteria  in  some  shape  or  form.  Hence  each  is  a  bacterial  treat- 
ment of  sewage.  It  may  appear  at  first  sight  that  such  a  process, 
involving,  as  it  does,  encouragement  to  the  growth  of  putrefactive 
bacteria,  is  not  without  danger.  But  we  shall  be  satisfied  that  this 
is  not  really  so,  when  it  is  remembered  that  the  bacterial  treatment 
of  sewage  is  under  control,  and  may  be  regulated  at  will.  Moreover, 
the  processes  of  decomposition  and  nitrification  ultimately  destroy 
the  pabulum  upon  which  the  organisms  in  question  depend  for  their 
existence,  and  hence  lead  to  their  death  when  they  have  fulfilled 
their  function. 

Two  applications  of  this  principle  have  long  been  in  vogue, 
namely,  the  intermittent  downward  jiltration  and  broad  irrigation. 


SEWAGE  DISPOSAL  161 

The  former  may  be  defined  as  "  the  concentration  of  sewage  at  short 
intervals,  on  an  area  of  specially-chosen  porous  ground  as  small  as 
will  absorb  and  cleanse  it,  not  excluding  vegetation,  but  making  the 
product  of  secondary  importance"  (Metropolitan  Sewage  Commis- 
sion). The  intermittency  is  essential,  and  the  process  is  partly 
mechanical  and  partly  bacterial,  that  is  to  say,  due  in  part  to  the 
nitrification  set  up  by  the  bacteria  in  the  superficial  layers  of  soil. 
For  successful  filtration  a  porous  soil  is  requisite,  a  proper  inclination 
of  the  land  to  allow  of  distribution,  and  a  division  into  areas,  in 
order  that  each  part  may  receive  sewage  for,  say,  six  hours,  and  then 
have  eighteen  hours'  rest.  Soil  pipes  carry  off  the  effluent.  Broad 
irrigation  (sewage-forms)  is  the  "  distribution  of  sewage  over  a  large 
surface  of  ordinary  agricultural  ground,  having  in  view  a  maximum 
growth  of  vegetation  (consistently  with  due  purification)  for  the 
amount  of  sewage  supplied/'  To  ensure  success,  the  area  must  be 
large  (say,  about  one  acre  to  every  100  of  the  population),  the  sewage 
passed  on  intermittently  to  allow  of  aeration  of  the  soil,  and  the  soil 
itself  must  be  light  and  porous.  Like  the  former,  there  is  a  bacterial 
influence  at  work  here.  Both  of  these  methods  are  much  to  be 
preferred  to  chemical  treatment  (and  were  recommended  by  the 
Sewage  Commission  of  1865) ;  yet,  on  account  of  space  and  manage- 
ment, as  well  as  on  account  of  the  tendency  of  the  land  to  clog  or 
become,  as  it  is  termed,  "  sewage  sick,"  their  success  has  not  been 
all  that  could  be  desired. 

In  1868,  a  Commission  was  appointed  to  inquire  into  the  best 
means  of  preventing  the  pollution  of  rivers.  They  made  several 
reports,  the  fifth  and  last  being  made  in  1874.  The  opinion  of  this 
Commission  on  the  comparative  merits  of  the  three  classes  of  pro- 
cesses for  the  treatment  of  sewage,  viz  : — chemical  precipitation, 
intermittent  filtration,  and  broad  irrigation,  may  be  stated  thus : — (1) 
All  these  processes  are  to  a  great  extent  successful  in  removing  pollut- 
ing organic  matter  in  suspension.  But  intermittent  filtration  is  best, 
broad  irrigation  ranks  next,  and  the  chemical  precipitation  processes 
are  less  efficient.  (2)  But  for  removing  organic  matters  in  solution  the 
processes  of  downward  intermittent  filtration  and  broad  irrigation  are 
greatly  superior  to  upward  filtration  and  chemical  processes. 

The  last  Commission  was  appointed  in  1882.  They  were  directed 
to  inquire  into  and  report  upon  the  system  under  which  sewage  was 
discharged  into  the  Thames  by  the  Metropolitan  Board  of  Works, 
whether  any  evil  effects  resulted  therefrom,  and  if  so,  what  measures 
could  be  applied  for  remedying  or  preventing  the  same.  In 
November  1884  they  issued  their  final  Eeport.  They  found  that 
evils  did  exist  "  imperatively  demanding  a  prompt  remedy,"  and  that 
by  chemical  precipitation  a  certain  part  of  the  organic  matter  of  the 
sewage  would  be  removed.  They  reported,  however,  "  that  the  liquid 

L 


162  BACTERIAL  TREATMENT  OF  SEWAGE 

so  separated  would  not  be  sufficiently  free  from  noxious  matters  to 
allow  of  its  being  discharged  at  the  present  outfalls  as  a  permanent 
measure.  It  would  require  further  purification,  and  this,  according 
to  the  present  state  of  knowledge,  can  only  be  done  effectually  by  its 
application  to  the  land." 

The  present  Eoyal  Commission  has  recorded  its  view  in  a  pre- 
liminary report  on  land  treatment, "  that  peat  and  stiff  clay  lauds  are 
generally  unsuitable  for  the  purification  of  sewage,  that  their  use  for 
this  purpose  is  always  attended  with  difficulty,  and  that  where  the 
depth  of  top  soil  is  very  small,  say  six  inches  or  less,  the  area  of  such 
lands  which  would  be  required  for  efficient  purification  would  in  cer- 
tain cases  be  so  great  as  to  render  land  treatment  impracticable."  On 
the  subject  of  effluents  they  state  in  the  same  preliminary  report  :— 

"  We  may,  however,  even  at  this  stage,  point  out  that  as  a  result 
of  a  large  number  of  examinations  of  effluents  from  sewage  farms  and 
from  artificial  processes,  we  find  that  while  in  the  case  of  effluents 
from  land  of  a  kind  suitable  for  the  purification  of  sewage,  there  are 
fewer  micro-organisms  than  in  the  effluents  from  most  artificial  pro- 
cesses, yet  both  classes  of  effluents  usually  contain  large  numbers  of 
organisms,  many  of  which  appear  to  be  of  intestinal  derivation,  and 
some  of  which  are  of  a  kind  liable,  under  certain  circumstances  at 
least,  to  give  rise  to  disease. 

"We  are  of  opinion,  therefore,  that  such  effluents  must  be 
regarded  as  potentially  dangerous,  and  we  are  considering  whether 
means  are  available  and  practicable  for  eliminating  or  destroying 
such  organisms,  or,  at  least,  those  giving  rise  to  infectious  diseases." 

Until  comparatively  recent  years,  the  above  methods  of  treating 
sewage  were  the  only  ones  available,  or,  at  all  events,  practised.  But 
now,  as  is  well  known,  some  new  applications  of  the  biological  treat- 
ment of  sewage  have  been  introduced,  which  call  for  consideration. 
Their  popularity  has  been  due  to  it  being  possible  to  adopt  them 
where  suitable  soil  did  not  exist  for  the  other  biological  methods,  and 
to  the  fact  they  have  been  on  the  whole  less  expensive  to  work. 
These  new  departures  depend  upon  bacteria  contained  in  the  sewage. 
The  process  may  depend  mainly  upon  anaerobes  (Cameron's  Septic 
Tank  or  Scott-Moncrieff's  process)  or  aerobes  (Duckett's  filter  or 
Dibdin's  filter).  These  may  be  conveniently  dealt  with  in  more  or 
less  chronological  order. 

The  Bacterial  Treatment  of  Sewage 

In  1872  the  Berlin  Sewerage  Commission  reported  that  sewage 
matter  was  converted  into  nitrates,  not  simply  by  molecular  processes 
but  by  organisms  present  in  sewage  and  soil.  Muntz,  Mueller,  and 
others  demonstrated  this  in  various  ways.  Mueller,  indeed,  had  shown 


PIONEER  WORK  163 

this  to  be  so  in  1865,  naming  spirilla,  vibrios,  and  a  "  protococcus  "  as 
the  organisms  in  question.  Then  in  1881  M.  Louis  Mouras,  of  Vesoul 
(Haute-Saone),  published  an  account  of  an  hermetically-sealed,  in- 
odorous, and  automatically  discharging  cesspool,  in  which  sewage  was 
anaerobically  broken  down  by  "  the  mysterious  agents  of  fermenta- 
tion." The  effluent,  a  homogeneous  and  scarcely  turbid  fluid,  was 
produced  in  a  tolerably  short  time  and  without  any  addition  of 
chemical  ingredients.  It  was  surmised  that  the  agents  of  fermenta- 
tion might  possibly  be  the  "anaerobes  of  M.  Pasteur."  This,  it  would 
seem,  is  the  first  record  we  have  of  the  treatment  of  sewage  by  simply 
allowing  Nature  to  fulfil  her  function  by  means  of  bacteria. 

The  next  step — and  indeed  as  regards  the  problem  in  England  the 
first  step — in  the  new  bacterial  treatment  of  sewage  was  inaugurated 
by  the  workers  (Jordan,  and  others)  under  the  State  Board  of  Health 
of  Massachusetts,  who  have  carried  out  a  laborious  series  of  experi- 
ments upon  sewage  purification  during  the  last  fifteen  years.  The  work 
undertaken  at  this  station  may  be  briefly  divided  into  three  main 
classes:  first,  purification  of  unfiltered  crude  sewage  by  means  of 
intermittent  filtration  through  sand  filters ;  secondly,  rapid  filtration 
of  sewage,  from  which  a  certain  amount  of  sludge  has  been  removed, 
by  different  methods  and  through  different  materials ;  thirdly,  purifi- 
cation by  dependence  upon  rapid  oxidation  or  burning  of  sludge 
either  by  forced  aeration  or  other  method  of  introducing  air  into  the 
filter.  Various  methods  have  also  been  devised  with  the  object  of 
getting  rid  of  the  insoluble  matter  in  sewage.  The  result  of  this 
extremely  valuable  work  by  the  Massachusetts  Board  clearly  demon- 
strated the  accuracy  of  the  fundamental  principle  that  on  prepared 
beds  or  "intermittent  downward  filtration"  the  contained  bacteria 
were  the  potent  agency.* 

Whilst  this  work  opened  up  a  new  prospect  for  the  bacterial 
treatment  of  sewage,  it  still  left  the  question  in  an  experimental  stage, 
and  then  it  was  that  Scott-Moncrieff,  Dibdin,  Cameron,  Ducat,  and 
others  carried  forward  the  work.f  It  was  in  1892  that  Mr  Scott- 

*  In  his  evidence  before  Lord  Brarawell's  Commission,  1883,  Dr  Sorby  had 
pointed  out  that  the  destruction  of  the  organic  sewage  matter  in  the  Thames  was  due 
to  bacteria,  and  that  it  was  only  when  these  were  unable  to  exert  their  functions  to 
the  full  extent  by  reason  of  deficient  aeration  of  the  water  that  putrefaction  set  in. 
t  The  following  general  classification  will  serve  to  show  the  nature  of  the  pro- 
cesses adopted  by  various  workers  : — 

Closed  septic  tank  and  contact  beds. 

Open  septic  tank  and  contact  beds. 

Chemical  treatment,  subsidence  tanks,  and  contact  beds. 

Subsidence  tanks  and  contact  beds. 

Contact  beds  alone. 

Closed  septic  tank  followed  by  continuous  filtration. 

Open  septic  tank  followed  by  continuous  filtration. 

Chemical  treatment,  subsidence  tanks,  and  continuous  filtration. 

Subsidence  tanks  followed  by  continuous  filtration. 

Continuous  filtration  alone. 


164  BACTERIAL  TREATMENT  OF  SEWAGE 

Moncrieff  introduced  his  cultivation  beds  filled  with  flint,  coke,  and 
gravel.  In  this  system  the  crude  sewage  passes  into  the  bottom  of 
the  bed,  the  liquid  portion  rises  through  the  bed,  and  the  suspended 
matter  is  kept  back  at  the  bottom,  where  it  undergoes  solution  by  the 
action  of  bacteria  present  upon  the  surfaces  of  the  flints,  etc.  In  order 
to  complete  the  process  highly  oxygenated  water  was  added  to  the 
effluent,  which  was  then  passed  down  a  "  nitrification  channel,"  where 
further  oxidation  was  secured.  The  results  of  this  process  were 
superior  to  anything  previously  obtained  in  this  country. 

Between  the  years  1891-95  Mr  Dibdin  experimented  with  sewage 
from  which  solid  organic  matter,  had  been  previously  removed  by 
screens  or  chemical  sedimentation.  Such  sewage  he  passed  through 
filter-beds  of  coke  breeze,  and  was  able  by  intermittent  filtration 
through  such  beds  (i.e.  allowing  rest  periods  between  charging  the 
filters)  to  obtain  a  purification  of  more  than  70  per  cent. 

Dr  Clowes  has  carried  on  this  work  on  behalf  of  the  London 
County  Council,  and  he  reports  that  the  sewage  is  allowed  to  flow 
into  large  tanks  which  contain  fragments  of  coke  about  the  size  of 
walnuts.  As  soon  as  the  level  of  the  liquid  has  reached  the  upper 
surface  of  the  coke-bed,  its  further  inflow  is  stopped,  and  it  is 
allowed  to  remain  in  contact  with  the  bacteria  coke  surface  for  about 
three  hours.  It  is  then  allowed  to  flow  slowly  away  from  the 
bottom  of  the  coke-bed.  After  an  interval  of  about  seven  hours,  the 
processes  of  emptying  and  filling  the  coke-bed  are  repeated  with  a 
fresh  portion  of  sewage.  The  coke-bed  is  usually  filled  in  this  way 
twice  in  every  twenty-four  hours.  The  aeration  of  even  the  lowest 
portions  of  a  deep  coke-bed  seems  to  be  satisfactory  in  the  above 
method  of  working,  since  the  air  present  in  the  interstices  of  the 
coke,  between  two  fillings  with  sewage,  usually  contains  75  per  cent, 
of  the  amount  of  oxygen  present  in  the  open  air. 

In  dealing  with  the  sewage  of  the  metropolis,  Dr  Clowes  holds 
that  it  is  best  to  allow  the  roughly-screened  raw  sewage  to  undergo  a 
somewhat  rapid  process  of  sedimentation,  in  order  to  permit  these 
matters  to  subside ;  and  then  to  pass  the  sewage  direct  into  the  coke- 
beds.  The  dissolved  matters  and  the  small  amount  of  suspended 
matters  which  are  still  present  in  the  sewage  are  then  readily  dealt 
with  by  the  bacteria  of  the  coke-bed,  and  practically  no  choking  of 
the  bed  occurs.  The  sewage  effluent  from  the  coke-bed  is  entirely 
free  from  offensive  odour,  and  remains  inoffensive  and  odourless  even 
after  it  has  been  kept  for  a  month. 

The  chemical  character  of  this  effluent  may  be  briefly  indicated 
by  stating  that  on  an  average  51 '3  per  cent,  of  the  dissolved  matter 
of  the  original  sewage,  which  is  oxidisable  by  permanganate,  has 
been  removed  by  the  bacteria,  and  that  the  portion  which  has 
been  removed  is  evidently  the  matter  which  would  become  rapidly 


SEPTIC  TANK  METHOD  165 

offensive,  and  would  rapidly  lead  to  deoxygenation  of  the  river  water 
if  it  were  allowed  to  pass  into  the  river.  The  above  percentage 
removal  (51*3)  was  effected  by  coke-beds  varying  from  4  to  6  feet  in 
depth.  A  similar  bed,  13  feet  in  depth,  has  proved  more  efficient, 
and  has  for  some  time  produced  a  percentage  purification  of  64  per 
cent.,  while  an  old  bed,  6  feet  in  depth,  has  given  a  percentage 
purification  of  86  per  cent.  A  repetition  of  the  treatment  of  the 
effluent  in  a  second  similar  coke-bed  has  produced  an  additional 
purification  of  19 '3  per  cent.,  giving  a  total  purification  of  70 '6  per 
cent.  Effluents  from  chemical  treatment  would  show  a  total 
purification  of  under  20  per  cent.  It  should  be  noted  that  the  above 
purification  is  reckoned  on  the  dissolved  impurity  of  the  sewage ; 
the  suspended  solid  matter  is  not  taken  into  account.  The  bacterio- 
logical condition  of  the  effluent  corresponds  in  the  main  with  that 
of  the  raw  sewage.  The  total  number  of  bacteria  undergoes  some 
reduction  in  the  coke-beds,  but  the  different  kinds  of  bacteria  which 
were  present  in  the  sewage  are  still  represented  in  the  effluent. 

From  these  and  many  other  similar  experiments,  it  has  come  to 
be  understood  that  the  bacterial  purification  depends,  as  we  have 
seen,  upon  two  main  groups  of  organisms,  namely,  those  that  are 
able  to  break  down  and  liquefy  solid  organic  matter,  and  those  that 
deal  with  it  when  in  solution.  Of  the  former  group,  some  act  best 
under  anaerobic  conditions.  No  strict  line  of  demarcation  can  be 
drawn  as  to  where  one  group  begins  and  the  other  absolutely  ends. 
It  is  a  complex  co-operation,  shared  in  by  a  large  variety  of 
organisms  classified  roughly  into  these  two  groups.  Systems  may 
be  introduced  in  which  the  anaerobes  are  encouraged  (as  at  Exeter), 
or  systems  may  be  established  on  an  aerobic  basis  (as  at  Sutton  and 
Manchester).  Hence  it  may  be  accepted  as  finally  settled  that  the 
bacterial  treatment  may  be  mainly  an  anaerobic  one  (Cameron,  Scott- 
Moncrieff,  and  others),  or  mainly  an  aerobic  one  (Dibdin,  Fowler,  and 
others),  or  a  mixture  of  the  two.  Whatever  system  is  used,  the  two 
great  agencies  of  breaking  down  and  oxidation  must  be  allowed 
ample  opportunity.  Probably  we  shall  most  clearly  recount  the 
application  of  these  principles  by  considering  in  some  detail  two 
examples  of  the  two  typical  methods  of  bacterial  treatment.  These 
two  examples  are  furnished  in  Cameron's  Septic  Tank  Installation 
(anaerobic),  and  at  the  Davyhulme  Works,  Manchester,  in  the 
Multiple  Contact  Bacteria-beds  (aerobic). 

1 .  Septic  Tank  and  Cultivation-bed  Method  (Cameron). — This 
method  has  been  adopted  at  Exeter  and  other  places.  The  plant  is 
twofold,  namely,  a  septic  tank  and  several  cultivation  or  bacteria  beds. 

The  septic  tank  is  a  large  underground  vault  constructed  of 
concrete,  cemented  on  exposed  surfaces,  and  having  a  capacity  of 
thousands  of  gallons,  according  to  the  population.  That  at  Exeter 


166  BACTERIAL  TREATMENT  OF  SEWAGE 

has  a  capacity  of  53,800  gallons,  and  takes  the  average  sewage  of 
1500  inhabitants  in  twenty-four  hours.  Near  the  entrance  is  a 
submerged  wall,  forming  a  grit  chamber  for  the  arrest  of  gravel 
and  coarser  detritus.  The  remaining  solid  matter  passes  into  the 
tank  itself.  The  inlet  and  outlet  being  below  the  level  of  the 
sewage,  light  and  air  are  excluded  as  far  as  possible.  Both  in  the 
sediment  at  the  bottom  of  the  tank  and  in  the  thick  scum  on  the 
surface  the  organic  compounds  are  broken  down  and  made  soluble. 
In  the  former  position  this  is  accomplished  by  anaerobic  bacteria,  in 
the  latter,  on  the  surface,  by  aerobic  bacteria.  It  need  hardly  be 
added  that  these  are  denitrifying  and  putrefactive  bacteria,  and  that 
those  at  the  bottom  of  the  tank  perform  greater  service  than  those  at 
the  top.  What  are  the  changes  taking  place  in  the  tank?  On 
every  side  throughout  the  tank  innumerable  small  masses  of  organic 
matter  may  be  observed  rising  and  falling.  At  first  the  masses  fall 
to  the  bottom  by  gravity ;  here  they  are  attacked  by  countless  bacteria 
which  generate  numerous  gases  in  the  small  masses  which  are  thus 
caused  to  rise  again  to  the  surface ;  the  pressure  being  then  reduced, 
the  gases  expand  and  burst  in  bubbles,  leaving  the  particles  to  sink 
again  and  commence  a  similar  cycle.  Thus  the  sewage  is  rapidly 
broken  down  by  a  process  of  peptonisation  and  digestion  (anaerobic 
hydrolysis)  until  all  the  organic  matter  is  in  solution  (soluble  nitro- 
genous compounds,  phenol  derivatives,  gases,  ammonia,  nitrites,  etc.). 
No  rest  is  necessary,  for  the  supply  of  organisms  is  unlimited,  being 
perpetually  replenished  by  incoming  sewage.  It  is  contended,  and 
probably  with  some  truth,  that  most  pathogenic  organisms  would  not 
be  able  to  survive  in  the  competition  which  must  be  present  in  the 
septic  tank.  When  the  liquid  sewage  passes  out  of  the  tank,  it 
differs  from  the  crude  sewage  entering  the  tank,  in  the  following 
particulars : — (a)  The  gravel  and  particular  debris  have  been  removed ; 
(&)  the  organic  solids  in  suspension  are  so  greatly  diminished  that 
they  are  almost  absent ;  (c)  there  is  an  increase  of  organic  matter  in 
solution ;  (d)  the  sewage  is  darker  in  colour  and  more  opalescent ; 
(e)  compounds  like  albuminoids,  urea,  etc.,  have  been  more  or  less 
completely  broken  down,  reappearing  in  more  elementary  conditions, 
like  ammonia,  methane,  carbonic  acid  gas,  and  sulphuretted  hydrogen. 
These  latter  bodies  may  be  in  solution,  or  may  have  escaped  as  gas. 

The  cultivation  beds  are  five  or  six  "  filters,"  to  which  the  sewage 
from  the  tank  flows,  and  by  an  automatic  arrangement  is  distributed 
to  each  bed  in  turn.  Each  filter  may  thus  be  full,  say,  about  six 
hours,  and  has  from  ten  to  twelve  hours'  rest.  The  depth  of  the 
filtering  medium  is  4  or  5  feet,  and  is  composed  as  follows  from  the 
bottom  upwards : — 

(a)  About  1  foot  in  depth  of  broken  furnace  clinker,  which  will 
pass  a  3-inch  mesh,  but  not  a  1-inch. 


SEPTIC  TANK  METHOD 


167 


168  BACTERIAL  TREATMENT  OF  SEWAGE 

(&)  Two  feet  or  so  of  screened  clinkers  to  pass  a  f-inch  mesh, 
but  not  a  J-inch  mesh. 

(c)  Three  inches  of  residue  from  above,  which  will  pass  a  ^-inch 
mesh. 

Collecting  drains  are  laid  on  the  bottom  of  the  beds,  joining  main 
collectors,  which  terminate  in  discharging  wells.* 

The  change  occurring  in  these  bacteria  beds  is  of  the  nature  of 
oxidation,  with  the  result  that  the  proportion  of  oxidised  nitrogen 
increases  (as  nitrites  and  nitrates),  the  ammonia  becomes  less  and 
the  total  solids  and  organic  nitrogen  almost  disappear.  It  will  thus 
be  seen  that  the  work  of  these  "  filters "  is  not  merely  a  straining 
action.  It  is  true  that  particulate  matter  in  the  effluent  from  the 
tank  is  caught  on  the  surface  by  the  film  (resulting  from  previous 
effluents),  but  the  real  work  of  the  bed  is  nitrification,  an  oxidation 
of  ammonia  into  nitrites  and  nitrates.  This  change  obviously  begins 
when  the  tank  effluent  flows  over  on  to  the  beds  and  the  oxygen 
then  obtained  by  the  effluent  is  carried  down  in  solution  into  the 
coke  breeze.  Upon  the  surfaces  of  the  filtrant  are  oxidising  bacteria. 
When  the  effluent  is  on  the  bed  they  oxidise  its  contained  products  ; 
when  the  bed  is  empty  and  "resting"  they  oxidise  carbon.  An 
advantage  arising  from  the  periodical  emptying  and  filling  of  the 
"  filter  "  is  that  the  products  of  decomposition  which  would  eventually 
inhibit  the  action  of  the  aerobic  bacteria  are  washed  away  and  pass 
into  the  nearest  stream,  or  on  to  the  land  direct,  where  they  become, 
of  course,  absolutely  innocuous.  The  "  filter "  is  perhaps  more 
correctly  termed  a  cultivation  bed,  for  its  purpose  is  to  furnish  a 
very  large  surface  upon  which  nitrifying  organisms,  present  as 
we  have  seen  in  all  soils,  may  flourish,  and  these  feeding  upon 
the  organic  matter  of  the  sewage,  may  perform  their  function  of 
oxidation. 

The  solid  matter  has  plenty  of  time  to  settle  in  the  tank  and  be 
fully  operated  on  by  the  bacteria,  which  are  not  only  contained  in 
the  sewage,  but  also  grow  and  multiply  in  the  tanks.  This  growth 
is,  of  course,  a  question  of  time;  and  just  as  the  growth  of  the 
nitrifying  layer  is  necessary  to  a  water  filter-bed,  so  the  growth  of 
the  necessary  organisms  is  required  in  the  septic  tank  and  on  the 
filter-beds.  In  the  tank,  however,  no  rest  is  necessary,  for  the  supply 
of  organisms  is  continuous  and  unlimited,  both  in  supply  and  in 
reproduction.  Not  so  the  beds.  Here  there  is  only  a  limited 
amount  of  oxygen  to  start  with,  and  consequently  a  definite  limita- 
tion to  the  amount  of  work  the  filters  can  perform.  Hence  the  need 
of  rest,  in  order  that  the  oxygen  may  be  replenished. 

The  amount  of  sludge  in  the  chemical  processes  has  always  been 

*  An  account  of  the  Septic  Tank  method  will  be  found  in  the  Brit.  Med.  Jour. , 
1900,  i.,  pp.  83-86. 


SEPTIC  TANK  METHOD 


169 


a  difficulty.    In  the  bacterial  processes  it  is  reduced  to  one-third,  and 
often  is  so  little  as  to  be  a  negligible  quantity. 

It  is  not  possible  to  lay  down  exact  limits  as  to  where  denitrifica- 
tion  ends  and  oxidation  begins.  To  a  certain  extent,  and  in  varying 
degree,  they  overlap  each  other.  But,  generally  speaking,  we  may 
say  that  in  the  tank  there  is  a  breaking-down  (denitrification  and 
decomposition)  and  in  the  filter-beds  a  building-up  (nitrification). 
The  case  is  precisely  parallel  to  similar  changes  occurring  in  soil, 


-Filter  Bed  full  of 
Burnt  Clay 

Exit  Pipe 


Filter  Bed  empty 

of  Filtrant,  showing 

"Exit  Pipe  at  the 

bottom 


FIG.  22.— Contact  Beds  (as  used  at  Sutton). 

and  with  which  we  have  already  dealt.  It  is  hardly  necessary  to  add 
that  there  is  a  marked  reduction  in  the  number  of  bacteria  present 
in  the  crude  sewage,  and  the  tank  and  cultivation-bed  effluents.  One 
investigation  has  shown  that  a  sample  of  crude  sewage  contained 
4,084,827  bacteria  per  c.c. ;  the  sewage  precipitate,  1,344,925 ;  the 
tank  effluent,  398,695  ;  and  the  cultivation-bed  effluent,  45,755  bacteria 
per  c.c. 

2.  Multiple    Contact    Bacteria   Beds.— This    method   in    its 
simplest  form  has  been  applied,  for  instance,  at  Sutton,  and  in  a 


170  BACTERIAL  TREATMENT  OF  SEWAGE 

more  advanced  form  at  Davyhulme,  Manchester.  At  Sutton  there  is 
no  tank.  A  metal  screen  holds  back  part  of  the  solids  from  being 
carried  on  to  the  beds.  The  filtrant  is  burnt  clay,  and  it  is  forked 
over  occasionally  to  let  in  oxygen.  The  beds  are  3J  feet  deep.  The 
bottom  of  the  bed  is  provided  with  a  6 -inch  main  drain  with  tributary 
drains.  The  crude  sewage,  after  passing  through  a  roughing  screen 
to  intercept  floating  paper,  etc.,  is  run  directly  upon  the  filter 
without  the  addition  of  any  chemicals.  The  filter  is  charged  to 
within  6  inches  from  the  surface,  and  the  sewage  remains  in  contact 
for  a  period  of  two  hours,  after  which  the  outlet  valve  is  opened  and  the 
filtrate  is  drawn  off  to  be  further  purified  on  fine-grain  bacteria  beds, 
after  which  the  effluent  is  in  a  fit  condition  to  be  discharged  into  the 
stream,  and  is  uniformly  superior  to  the  effluent  obtainable  by 
chemical  treatment.  The  sludge  is  absorbed  by  bacterial  agency  in 
the  beds,  and  does  not  accumulate  or  manifest  itself.  The  beds  are 
free  from  any  offensive  odour.  At  first  the  beds  were  seeded  with 
Micrococcus  candicans,  but  it  is  now  known  that  the  necessary  bacteria 
are  in  the  sewage,  and  seeding  is  not  required.  For  more  effective 
screening  of  the  sewage  an  automatic  rotary  screen  may  be  fixed. 
This  screen  may  be  driven  by  a  Poucelet  water-wheel,  actuated  by 
the  sewage. 

Experiments  seem  to  prove  that  coarse-grain  beds  worked  on  the 
contact  principle  may  be  constructed  of  a  numerous  class  of  materials, 
and  that  different  districts  may  adopt  materials  which  are  obtainable 
locally,  and  often  at  a  small  cost,  although  it  may  be  observed  that 
porous  coarse-grained  materials  such  as  coke  and  burnt  ballast  effect 
a  greater  degree  of  purification  than  do  fine-grained  impervious 
material  such  as  granite,  slate,  etc.  The  cost  of  such  a  system  would 
be  in  many  cases  one-quarter  of  a  chemical  precipitation  and 
irrigation  system,  and  yet  more  effective.  It  will  be  understood  that 
the  absence  of  a  septic  tank  does  not  mean  an  entire  absence  of  the 
anaerobic  action  of  the  process.  It  simply  takes  for  granted  that 
this  portion  of  the  process  has  been  in  part  performed  in  the  sewers. 

The  Manchester  bacterial  system  is  practically  the  same  in 
principle  as  at  Sutton.  But  there  are  two  important  and  interesting 
differences.  First,  the  quantity  of  sewage  to  be  dealt  with  is  very 
much  greater.  Secondly,  there  is  the  added  complication  that  the 
Manchester  sewage  contains  large  quantities  of  tfnz^e-effluent  from 
breweries,  dyeing  and  bleaching  works,  galvanising  works,  tanneries, 
and  derivatives  from  coal-tar,  colour,  and  naphthalene  works.  It  has 
been  frequently  suggested  that  such  chemicals  in  the  sewage  would 
prevent  the  contained  bacteria  from  fulfilling  their  rdle  in  the  purifi- 
cation. Consequently,  the  trial  of  the  two  chief  methods  of  bacterial 
treatment  of  Manchester  sewage  has  been  followed  with  much 
interest.  In  1898  three  experts  were  appointed,  and  requested  to 


CONTACT  BEDS  171 

furnish  a  report  as  to  the  system  best  adapted  for  Manchester. 
Various  points  demanded  elucidation  which  had  previously  escaped. 
These  were  chiefly  (1)  whether  trade-refuse  in  the  sewage  impaired 
the  efficiency  of  bacterial  purification ;  (2)  whether  a  portion,  at  any 
rate,  of  the  sludge  can  be  destroyed  by  bacterial  agency ;  (3)  whether 
chemical  precipitation,  as  in  Dibdin's  first  method,  before  bacterial 
treatment  could  be  dispensed  with ;  (4)  whether  an  aerobic  process 
alone  or  a  combination  of  anaerobic  and  aerobic  processes  is  the  more 
effective.  With  these  objects  in  view  a  septic- tank  (Cameron) 
method  and  a  filter-bed  method  were  installed,  and  under  the  super- 
intendence of  Dr  G-.  J.  Fowler,  the  superintendent  and  chemist  of 
the  Corporation  Sewage  Works,  the  observations  were  carried  out.* 

That  the  experts'  view  of  the  bacterial  treatment  of  sewage  was 
similar  to  that  set  forth  above  may  be  gathered  from  a  preliminary 
note:  "The  bacteria  already  existing  in  the  sewage,"  they  state, 
"  are  brought  by  it  on  to  the  bacteria  beds,  or  into  the  septic  tank. 
The  former,  by  providing  an  enormously  extended  surface  for  the 
development  of  bacterial  growths,  furnish  an  ideal  habitat  for  the 
aerobic  micro-organisms  which  require  air  for  the  display  of  their 
powers,  whilst  the  septic  tank,  by  confining  a  large  volume  of  liquid 
which  is  but  superficially  in  contact  with  air,  enables  the  anaerobic 
micro-organisms  to  work  to  great  advantage.  It  will  be  understood 
that  some  time  must  elapse  before  the  bacterial  life  attains  its 
maximum,  either  in  the  bacteria  bed  or  in  the  septic  tank,  and 
consequently  the  amount  of  sewage  which  can  be  purified  therein 
will  gradually  increase  as  time  goes  on."-f- 

The  contact  beds  at  Manchester  were  five  in  number,  and  of  different 
area.  In  principle  they  were  of  similar  construction  to  those  described 
already.  The  filtering  medium  was  clinkers  laid  to  a  depth  of  3  feet, 
and  of  varying  size  in  the  five  beds,  but  uniform  in  each  bed. 
Clinkers  which  passed  f-inch  mesh,  rejected  by  J-inch  mesh,  were 
found  to  be  the  best  grade.J 

Arrangements  are  made  by  which  it  is  possible  to  admit  sewage 
to  the  beds  in  three  different  conditions,  namely,  raw,  screened 
sewage ;  sewage  which  has  undergone  settlement  in  a  small  settling 
tank ;  sewage  which  has  undergone  both  settlement  and  anaerobic 
action.  Eventually  a  plan  was  adopted  by  which  the  sewage  was 

*  A  full  record  of  the  work  done  at  Manchester  will  be  found  in  the  Rivers 
Department  Reports  for  1901,  1902,  1903,  and  1904. 

f  City  of  Manchester,  Rivers  Department.  Experts'  Report  on  Treatment  of 
Manchester  Sewaye,  1899,  p.  12.  (Mr  Baldwin  Latham,  Professor  Percy  Frankland, 
F.R.S.,  and  Professor  W.  H.  Perkin,  Junr.,  F.R.S.). 

J  It  appears  that  the  initial  capacity  of  a  contact  bed  is  uninfluenced  by  the 
grade  of  clinker.  At  first  there  is  a  rapid  decrease  in  capacity,  due  in  part  to 
sinking  of  the  surface  and  in  part  to  bacterial  growth  on  the  clinkers,  which  must 
necessarily  occupy  some  space,  even  though  relatively  little.  In  a  comparatively 
short  time  the  beds  acquire  a  constant  average  capacity. 


172  BACTERIAL  TREATMENT  OF  SEWAGE 

passed  through  a  settling  tank  prior  to  its  being  brought  on  to  the 
contact  beds.  In  short,  the  method  resolved  itself  into  one  of  an 
open  septic  tank  and  multiple  contact  following  the  settlement  or 
screening  out  of  grosser  solids.  The  results  surpassed  even  the  most 
sanguine  expectations,  even  though  the  beds  were  filled  four  times 
daily.  If  the  two  methods — namely,  the  closed  septic  tank  and 
single  contact,  and  the  open  tank  and  multiple  contact — are  com- 
pared, it  is  the  opinion  of  the  three  experts  named  that  "  where  both 
systems  are  dealing  with  the  same  volume  of  sewage  on  the  same 
area,  the  advantage  as  regards  efficiency  belongs  indisputably  to  the 
double  contact  system."  Boyce  subsequently  confirmed  this  conclusion 
in  favour  of  a  combination  of  the  anaerobic  and  aerobic  processes, 
provided  that  the  septic  process  was  perfected,  and  the  suspended 
sludge  did  not  pass  over  on  to  the  beds.* 

Before  summarising  the  main  conclusions  which  may  now  be 
legitimately  drawn  from  the  Manchester  experiments,  a  word  or  two 
may  be  said  concerning  the  characters  of  an  efficient  bacteria  bed 
and  the  management  of  storm-waters  in  sewage  treatment. 

The  material,  or  filtrant,  of  which  the  bed  is  composed  may  vary 
within  wide  limits.  Burnt  clay,  coke,  clinkers,  cinders,  or  various 
forms  of  gravel  may  all  be  efficient,  provided  there  is  ample  aeration 
and  porosity.  The  required  organisms  exist,  of  course,  mainly  in  the 
sewage,  but  they  require  abundant  oxygen  in  order  to  perform  their 
function.  To  assist  in  maintaining  this  aeration  the  surface  should 
be  raked  over  from  time  to  time.  It  has  been  suggested  that  in 
times  of  frost  a  layer  of  ice  would  prevent  the  action  of  the  bed. 
But  in  point  of  fact  such  obstruction  would  rarely  occur,  the 
temperature  of  the  sewage  being  sufficient  both  to  prevent  such 
stoppage  of  the  beds  and  also  to  maintain  the  necessary  activity  of 
the  bacteria,  which,  as  we  have  already  seen,  require  for  their  vitality 
nutriment,  oxygen,  moisture,  and  a  favourable  temperature.  From 
December  to  April  the  average  daily  temperature  of  the  Manchester 
sewage  was  55*5°  F.,  whilst  the  average  temperature  of  the  sur- 
rounding air  was  45  "3°  F.  Hence  the  ice  difficulty  is  naturally 
overcome.  Another  point  of  importance  in  connection  with  aeration 
is  the  allowance  of  sufficiently  frequent  and  prolonged  periods  of 
rest.  Without  such  intervals  the  beds  would  of  course  become 
clogged,  and  eventually  inactive,  because  lacking  in  aerobic  bacteria. 
Though  not  absolutely  a  character  of  the  beds,  there  is  one  further 
point  always  to  be  borne  in  mind  in  securing  their  efficiency.  It 
is,  that  the  sewage  being  applied  to  the  bed  should  be  as  far  as 
possible  uniform  in  consistence  and  freed  from  suspended  matters 
by  sedimentation.  Any  suspended  matter  not  so  removed  should  be 
retained  as  far  as  possible  on  the  surface  of  the  bed. 

*  Royal  Commission  on  Sewage  Disposal,  1902,  p.  11. 


CONTACT  BEDS  173 

A  moment's  reflection  will  make  it  evident  that  the  problem  may 
be  seriously  complicated  at  short  notice  by  the  great  increase  in 
volume  of  the  sewage  following  rain-storms.  To  this  matter  the 
experimenters  at  Manchester  have  also  directed  their  attention. 
They  draw  the  necessary  distinction  between  the  first  flush  of  a 
storm  and  the  highly-diluted  sewage  which  follows,  designating  the 
latter  only  as  "  storm  water."  They  decide  that  provision  must  be 
made  for  the  storage  or  separate  treatment  of  "  first  flush  "  of  sewage 
at  the  beginning  of  a  storm,  and  that  about  two  hours  after  the 
augmented  flow  is  the  time  to  commence  accelerated  treatment,  the 
exact  procedure  varying  according  to  the  character  and  duration  of 
the  storm.  Short  double  contacts,  or  even  a  single  contact,  is 
sufficient  to  purify  storm  water,  and  there  is  no  decrease  in  the 
purifying  capacity  of  the  bed. 

Summarily,  the  final  conclusions  arrived  at  by  Latham,  Frank- 
land,  and  Perkin  were  as  follows : — 

"  1.  That  the  bacterial  system  is  the  system  best  adapted  for  the 
purification  of  the  sewage  of  Manchester. 

"  2.  That  any  doubts  which  may  have  arisen  in  the  first  instance 
as  to  its  suitability,  owing  to  the  presence  in  Manchester  sewage  of 
much  manufacturing  refuse,  have,  through  the  convincing  results  of 
our  experimental  inquiry,  been  entirely  banished. 

"  3.  That  inasmuch  as  a  bacterial  contact  bed  can  only  effect  a 
definite  amount  of  purification  in  a  single  contact,  it  becomes 
necessary,  in  order  to  carry  the  purification  beyond  this  limit,  to 
apply  the  effluent  to  a  second  bed,  in  which  again  a  further  definite 
amount  of  purification  can  be  effected.  Hence,  for  obtaining  a  high 
degree  of  efficiency  in  bacterial  purification  of  sewage,  a  system  of 
multiple  contact  is  generally  necessary.  Thus  it  may  be  taken 
broadly  that  in  the  first  contact  50  per  cent,  of  the  dissolved  impurity 
is  removed,  and  that  in  the  second  contact  50  per  cent,  of  the 
impurity  still  remaining  in  the  effluent  is  disposed  of,  and  so  on."  * 

In  subsequent  experiments  these  conclusions  were  amply  con- 
firmed, and  the  Manchester  Corporation  eventually  extended  their 
sewage  works,  laying  down  five  additional  tanks,  and  a  large  number 
of  contact  beds  (primary  and  secondary).  These  included  92  half -acre 
primary  beds,  with  26  acres  storm-water  filter-beds  at  Davyhulme, 

*  Elvers  Department  of  Manchester,  Experts'  Report,  1899,  p.  53.  The 
Borough  Surveyor  of  Leicester  (Special  Report,  1900)  examined  various  bacterial 
systems  for  the  disposal  of  the  sewage  of  the  Belgrave  district,  and  finally 
recommends  as  the  best  method  the  following :  (1)  Crude  sewage  passed  into  an 
open  or  closed  detritus  tank  to  remove  suspended  mineral  matters  ;  (2)  then  on  to 
clarifying  bacteria  beds  of  4  feet  6  inches  depth,  and  containing  crushed  and 
screened  clinkers  (coarse  and  fine)  from  the  refuse  destructors  ;  three  fillings  a  day  ; 
(3)  finally,  land  purification  of  the  effluent  on  old  pasture.  (Total  purification  of 
suspended  matter  of  sewage  99 '12  per  cent.  ;  of  albuminoid  ammonia,  8676  per 
cent.  ;  and  of  oxygen  absorbed  91  '08  per  cent.). 


174  BACTERIAL  TREATMENT  OF  SEWAGE 

and  46  acres  of  secondary  contact  beds  on  land  at  Flixton.*  Dr 
Fowler,  the  superintendent  and  chemist,  concludes  that  the  bacterial 
process  is  best  conducted  in  three  stages: — (a)  Settlement  and 
screening  out  of  the  grosser  solids ;  (b)  Anaerobic  decomposition  in 
the  septic  tank ;  and  (c)  Oxidation  on  bacteria  beds.  He  concludes 
in  respect  of  the  septic  tanks : — 

That  the  effluents  from  closed  and  open  septic  tanks  are  practically  indentical  in 
composition,  and  that  with  a  tank  space  equal  to  half  the  daily  flow  of  Manchester 
sewage,  it  is  possible  to  digest  about  25  per  cent,  of  the  total  suspended  matter  in 
the  sewage.  The  suspended  matter  in  the  septic-tank  effluent  is  of  a  granular  char- 
acter, and  readily  separates  out  on  standing,  and  when  arrested  on  the  surface  of  a 
bacteria  bed  does  not  seriously  impede  the  free  flow  of  the  water  into  the  bed.  The 
organic  matter  in  solution  is  much  more  easily  nitrified  than  that  present  in  fresh 
sewage,  so  that  it  is  possible  with  one  contact  to  constantly  obtain  non-putrefactive 
filtrates.  The  blending  which  takes  place  in  the  septic  tank  is  of  value  in  minimis- 
ing the  effect  of  excessive  amounts  of  manufacturing  refuse,  and  in  producing  an 
effluent  of  fairly  constant  composition. 

In  respect  to  contact  beds  he  points  out  that  the  capacity  of  contact  beds  suffers 
a  rapid  initial  decrease,  but  afterwards,  with  careful  working,  the  rate  of  decrease  is 
very  much  less. 

The  causes  of  loss  of  capacity  appear  to  be  five,  namely — (a)  Settling  together  of 
the  material ;  (6)  Growth  of  organisms ;  (c)  Impaired  drainage ;  (d)  Insoluble 
matter  entering  bed ;  and  (e)  Breaking  down  of  material.  These  matters  will  be 
found  fully  discussed  in  the  annual  reports  of  the  Rivers  Department,  and  we  have 
not  space  to  enter  into  them  here.  We  may,  however,  briefly  refer  to  the  conditions 
of  the  successful  working  of  contact  beds  as  arrived  at  as  a  result  of  the  Manchester 
experience : — 

(1)  The  bed  should  be  worked  very  slowly  at  first,  in  order  to  allow  it  to  settle 
down  and  the  bacterial  growths  to  form.  In  this  way  there  will  be  less  danger  of 
suspended  matter  finding  its  way  into  the  body  of  the  bed,  while  the  material  is 
still  loose  and  open.  (2)  The  burden  should  not  be  increased  till  analysis  reveals 
the  presence  of  surplus  oxygen,  either  dissolved  or  in  the  form  of  nitrates  in  the 
effluent.  (3)  Analyses  of  the  air  in  the  bed  may  usefully  be  made  from  time  to  time 
during  resting  periods.  (4)  The  variations  in  capacity  should  be  carefully  recorded. 
If  the  capacity  is  found  to  be  rapidly  decreasing,  a  period  of  rest  should  be  allowed. 
(5)  Long  periods  of  rest  should  be  avoided  during  winter,  as  when  deprived  of  the 
heat  of  the  sewage  the  activity  of  the  organisms  decreases.  If  necessary,  the 
burden  on  the  bed  should  then  be  decreased  by  reducing  the  number  of  fillings  per 
day,  rather  than  by  giving  a  long  rest  at  one  time.  (6)  The  insoluble  suspended 
matter  should  be  retained  on  the  surface  by  covering  the  latter  with  a  layer  of  finer 
material  not  more  than  three  inches  in  depth.  The  suspended  matter  thus  arrested 
should  not  be  raked  into  the  bed,  but  when  its  amount  becomes  excessive  it  should 
be  scraped  off.  This  should  be  done  if  possible  in  dry,  warm  weather,  after  the  bed 
has  rested  some  days.  By  placing  the  inlet  and  outlet  penstocks  as  close  together 
as  possible,  the  suspended  matter  will  tend  to  concentrate  in  their  vicinity,  and  its 
removal  will  be  facilitated,  f 

How  far  the  various  applications  of  the  bacterial  agency  in  puri- 
fication will  pass  the  scrutiny  of  the  Koyal  Commission  on  Sewage 
Treatment,  now  sitting,  it  is  impossible  to  say.  But  there  can  be  no 

*  The  particulars  as  to  these  new  works,  their  construction,  materials,  capacities, 
etc.,  will  be  found  in  the  Manchester  Rivers  Department  Report,  1902,  with  plates 
and  charts,  pp.  18-24,  and  more  recent  extensions  in  subsequent  Reports  (1903-4). 

f  City  of  Manchester  Rivers  Department,  Annual  Report,  1902,  p.  15. 


RELATION  TO  DISEASE  ORGANISMS  175 

longer  any  doubt  that  some  form  of  such  agency  is  the  only  efficient, 
because  the  only  natural,  means  of  disposing  of  sewage. 

The  Effect  of  the  Bacterial  Treatment  upon  Disease- 
Producing*  Organisms 

It  has  been  urged  from  time  to  time  by  the  advocates  of  the  vari- 
ous methods  of  bacterial  treatment,  that  pathogenic  organisms  are 
destroyed  during  the  purification  in  many  of  these  processes.  It  is 
clearly  a  matter  of  importance  to  know  how  far  an  effluent,  in 
addition  to  being  non-putrescible  and  fully  nitrified,  also  possesses 
no  disease-producing  capacity.  We  have  already  seen,  from  the 
researches  of  Klein,  and  Laws  and  Andrewes,  that  sewage  is  not  a 
favourable  medium  for  B.  typJiosus.  The  bacillus  of  Asiatic  cholera 
is  known  to  be  but  little  less  favoured  by  sewage.  The  spread  of 
diphtheria  by  sewage  is  at  least  a  matter  of  doubt,  and  Shattock's 
experiments  tend  to  prove  that  in  any  case  the  virulence  of  the 
Bacillus  diphtherice  is  not  increased  by  sewer  air. 

Anything  like  exhaustive  researches  into  the  effect  of  the  septic 
tank  or  cultivation  beds  upon  pathogenic  germs  has  not  been  under- 
taken up  to  the  present,  and  we  can  only  conjecture  as  to  their  fate. 
Dr  Houston  has  made  a  cautious  declaration  upon  this  matter,  and  at 
present  we  have  not  evidence  to  justify  a  more  certain  statement. 
"  The  balance  of  evidence,"  he  says,  "  points  to  the  probability  that 
some,  at  all  events,  of  the  pathogenic  organisms  are  crowded  out  in 
the  struggle  for  existence  in  a  nutritive  medium  containing  a  mixed 
bacterial  flora,  their  vitality  being  weakened  or  destroyed  by  the 
enzymes  of  the  saprophytic  species."  *  He  further  adds :  "  It  must 
be  distinctly  understood  that  I  do  not  imply  that  such  organisms  as 
the  typhoid  bacillus  or  the  cholera  vibrio  would  necessarily  lose  their 
vitality,  or  even  suffer  a  diminution  in  virulence  under  the  conditions 
prevailing  in  a  biological  filter.  In  the  absence  of  actual  experi- 
ments, I  am  not  prepared  to  say  more  than  that  I  believe  that  if  these 
germs  did  gain  access  to  the  sewage  they  would  suffer  a  diminution 
in  numbers  primarily  in  the  sewers  [or  septic  tank],  and  secondarily 
in  the  coke-beds  [or  cultivation  beds]."  Subsequently,  as  a  result  of 
further  experience  of  the  effluent  from  the  Crossness  Sewage  Works, 
Houston  wrote,  "  However  satisfactory  the  process  may  be  from  the 
chemical  and  practical  point  of  view,  the  effluents  from  the  bacterial 
beds  cannot  reasonably  be  assumed  to  be  more  safe  in  their  possible 
relation  to  disease  than  raw  sewage." f 

Indirectly  connected  with  this  point,  a  word  or  two  may  be  added 
concerning  some  recent  investigations  made  by  Dr  Houston  upon  the 

*  Bacterial  Treatment  of  Crude  Sewage,  1899  (Second  Report),  p.  19. 
t  Edin.  Med.  Jour.,  Feb.  1901. 


176  BACTERIAL  TREATMENT  OF  SEWAGE 

deposit  which  accumulates  on  coke  fragments  used  in  the  beds  at 
Barking  and  Crossness.*  The  coke  was  found  to  be  coated  with  a 
black-coloured  slimy  deposit,  free  from  objectionable  smell,  and 
almost  odourless.  On  examination  of  the  deposit,  diluted  with 
sterile  water,  and  making  cultures,  it  was  found  that  the  number  of 
bacteria  per  gramme  of  the  deposit  was  1,800,000.  This  number, 
large  as  it  may  seem,  would  weigh  only  a  minute  fraction  of  a 
gramme,")*  so  that  it  is  evident  that  the  number  of  living  bacteria  do 
not  in  themselves  account  in  any  way  for  the  deposit.  As  to  the 
nature  of  these  organisms,  Dr  Houston  adds :  "  The  character  of  the 
microbes  appearing  in  the  cultures  differed  somewhat  from  those 
found  in  crude  sewage.  For  example,  there  was  an  increase  in  the 
number  of  spores  of  Bacillus  enteritidis  sporogenes  (Klein),  and  a 
decrease  in  the  number  of  B.  coli.  Proteus-like  germs  were  present 
in  abundance,  many  being  of  P.  mirabilis  type.  Further,  B. 
arborescens  and  an  allied  form  were  present  in  considerable  numbers. 
An  organism  apparently  identical  with  B.  prodigiosus  was  also 
isolated/'J  The  deposit  also  contained  a  number  of  bacilli  with  pre- 
cisely similar  staining  properties  as  those  of  tubercle  bacilli  (acid- 
fast).  They  were  also  morphologically  indistinguishable  from  the 
tubercle  bacillus.  In  one  instance  Houston  isolated  a  virulent 
tubercle  bacillus  from  a  sewage  effluent.  Such  facts  are  of  evident 
practical  importance  in  relation  to  the  final  disposal  of  the  effluent, 
whether  it  is  discharged  into  a  stream  used  for  drinking  purposes  or 
otherwise. 

Many  of  the  researches  having  for  their  object  the  fate  of 
pathogenic  organisms  in  sewage  have  been  based  upon  the  typhoid 
bacillus  as  a  type.  Laws  and  Andrewes,  in  addition  to  demonstrat- 
ing that  this  organism  could  only  live  in  sewage  a  short  time,  showed 
that  one  sewage  bacillus  {B.  fluorescens  stercoralis)  possessed  the  chief 
powers  of  antagonism,  and  it  is  probable  that  the  contained  bacteria 
rather  than  the  chemical  products  of  sewage  act  as  unfavourable 
conditions  for  the  typhoid  bacillus.  Horrocks  found  that  in  sterilised 
sewage  the  typhoid  bacillus  could  live  for  sixty  days.§  Houston  has 
thrown  light  upon  the  fate  of  B.  typhosus  by  his  work  on  the 
occurrence  of  B.  coli  and  B.  enteritidis  sporogenes  in  effluents,) |  and 
Miss  Chick  has  furnished  evidence  in  respect  of  B.  coli,  tending  in 
the  direction  of  showing  that  after  sewage  had  passed  over  double 
contact  beds  about  75  per  cent,  of  the  B.  coli  were  removed,  and 

*  Bacterial  Treatment  of  Crude  Sewage  (Supplement  to  Second  Report),  1899, 
p.  4. 

t  Dr  Houston  finds  that  1,800,000  typhoid  bacilli  weigh  only  0 '0000147 
gramme. 

J  Ibid.,  p.  4. 

§  Jour,  of  Sanitary  Institute,  January  1900. 

||  First,  Second,  and  Third  Reports  to  the  London  County  Council  (vide  supra}. 


PLATE  17. 


Bacillus  anthracis.    Smear  preparation  from  splenic  juice  of  guinea-pig  that  died  after  inoculation 
with.  2  c.c.  of  Yeovil  septic  tank  liquor,     x  500. 


Bacillus  anthracis.     "  Impression  "  preparation  from  a  surface  gelatine  plate  culture,  24  hours  at  20°  C. 
Stained  with  methylene  blue.       x  1000. 


[To  face  page  176. 


RELATION  TO  DISEASE  ORGANISMS  177 

after  land  filtration  practically  an  effluent  might  be  free  from  B. 
coli*  Pickard  has  recently  shown  that  the  typhoid  bacillus  vanishes 
from  crude  sewage  in  about  fourteen  days.  If  the  percentage  of 
original  amount  of  typhoid  bacillus  introduced  into  the  sewage  for 
experimental  purposes  be  100 ;  in  twenty-four  hours  it  has  fallen  to 
76  per  cent.,  in  thirty-two  hours  to  71  per  cent,  in  forty-eight  hours 
to  60  per  cent.,  in  seven  days  to  8  per  cent.,  and  in  fourteen  days  to 
0'73  per  cent.  He  also  demonstrated  that  a  large  proportion  (90 
per  cent.)  of  typhoid  bacilli  are  actually  destroyed  in  filter-beds  such 
as  are  used  in  the  bacterial  treatment  of  sewage,  f 

Houston  likewise  has  found  the  B.  anthracis  in  septic-tank 
liquor  and  sludge,  and  in  the  secondary  beds  and  general  effluent.  He 
also  found  the  anthrax  bacillus  in  the  mud  of  the  banks  of  the  river 
Yeo  at  Yeovil  within  150  feet  of  the  main  sewer.  The  spores  of 
anthrax  are  peculiarly  resistant,  and  it  is  in  this  form  that  the  bacillus 
can  pass  through  sewage  unaffected  {  (Plate  17).  The  same  author 
has  demonstrated  that  the  B.  pseudo-tuberculosis  of  Pfeiffer  may  be 
present  in  the  effluent  of  various  sewage  processes,  and  the  same  is 
true  of  B.  pyocyaneus  which,  however,  occurs  more  rarely.  Both 
these  organisms  are  highly  pathogenic  to  lower  animals,  and  are 
also  related  to  morbid  processes  occurring  in  the  human  subject. § 
Houston,  who  has  made  extended  inquiries  on  this  question  of  the 
effect  of  bacterial  treatment  of  sewage  on  pathogenic  organisms, 
summarises  his  conclusions  by  stating  that  biological  treatment  on 
land,  or  by  artificial  processes,  does  not  necessarily  remove  patho- 
genicity  from  the  sewage  effluent ;  that  the  absence  of  pathogenic 
result  when  sewage  has  been  filtered  shows  that  the  products  of 
pathogenic  bacteria  in  sewage  are  not  of  a  markedly  poisonous 
nature ;  and  that  the  pathogenicity  of  sewage  may  depend  on  spores 
rather  than  bacilli.  ||  These  conclusions  must,  however,  be  accepted 
with  reserve,  and  in  a  relative  sense  only,  at  present.  Broadly,  it 
may  be  said  that  if  sewage  contains  pathogenic  bacteria,  and  is  then 
treated  by  bacterial  methods,  the  effluent  cannot  be  certainly 
assumed  to  be  safer  in  this  respect  than  the  raw  sewage  slightly 
diluted ;  or,  expressed  in  other  words,  Houston's  work  indicates  "  the 
inadvisability  of  relying  on  septic  tanks,  contact  beds,  or  continuous 
filters  to  remove  altogether  the  element  of  potential  danger  to  health 
associated  with  the  discharge  of  effluents  from  these  processes  of 
sewage  treatment  into  drinking-water  streams."  IF 

*  Thompson- Yates  Laboratory  Report,  vol.  iii.,  part  i.,  1900. 

f  Jour,  of  State  Medicine,  1903,  pp.  203-210. 

J  Royal  Commission  on  Sewage  Disposal,  Second  Report,  1902,  p   39 

§  Ibid.,  p.  54.  ||  Ibid.,  p.  58. 

1T  Ibid.,  Fourth  Report,  1904,  vol.  iii.,  pp.  77-96. 

M 


CHAPTEE  VII 

BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

General  Principles— Sources  of  Pollution— Number  of  Bacteria  in  Milk — Influence 
of  Time  and  Temperature — Species  of  Bacteria  found  in  Milk — Fermentations 
of  Milk — Pathogenic  Organisms  in  Milk — Milk-borne  Disease  :  Tuberculosis, 
Typhoid  Fever,  Scarlet  Fever,  Sore-Throat  Illnesses,  Cholera,  Epidemic 
Diarrhoea — Preventive  Measures — Protection  of  Milk  Supply — Control  of 
Milk  Supply  :  Refrigeration,  Straining,  Sterilisation,  Pasteurisation— Special- 
ised Milk— Bacteria  in  Milk  Products— Cream-Ripening-Butter-Making— 
Cheese-Making — Abnormal  Cheese-Ripening—Poisonous  Cheese. 

INJURIOUS  micro-organisms  in  foods  are,  fortunately  for  the  con- 
sumers, usually  killed  by  cooking.  Vast  numbers  are,  as  far  as  we 
know,  of  no  harm  whatever.  Alarming  reports  of  the  large  numbers 
of  bacteria  which  are  contained  in  this  or  that  food  are  generally  as 
irrelevant  as  they  are  incorrect.  Bacteria,  as  we  have  seen,  are 
ubiquitous.  In  food  we  have  abundance  of  the  chief  thing  necessary 
to  their  life  and  multiplication — favourable  nutriment.  Hence  we 
should  expect  to  find  in  uncooked  or  stale  food  an  ample  supply  of 
saprophytic  bacteria.  There  is  much  wholesome  truth  in  the 
assertion  that  good  food  as  well  as  bad  frequently  contains  large 
numbers  of  bacteria,  and  often  of  the  same  species.  It  is  well  that 
we  should  become  familiarised  with  this  idea,  for  its  accuracy  cannot 
be  doubted,  and  its  acceptance  at  the  present  time  may  not  be  with- 
out beneficial  effect. 

Nevertheless,,  it  is  well  we  should  know  the  bacterial  flora  of 
good  and  bad  foods,  for  at  least  two  reasons.  First,  there  is  no  doubt 
whatever  that  a  considerable  number  of  cases  of  poisoning  can  be 
traced  every  year  to  food  containing  harmful  bacteria  or  their  products. 
To  several  of  the  more  illustrative  cases  we  shall  have  occasion  to 
refer  in  passing.  Secondly/  we  may  approach  the  study  of  tlin 
bacteriology  of  foods  with  some  hope  that  therein  light  will  be  found 

178 


GENERAL  PRINCIPLES  179 

upon  some  important  habits  and  effects  of  microbes.  There  can  be 
little  doubt  that  food-bacteria  afford  an  example  of  association  and 
antagonism  of  organisms  to  which  reference  has  already  been  made. 
Any  information  that  can  be  gleaned  to  illumine  these  abstruse 
questions  would  be  very  welcome  at  the  present  time.  But  there  is 
a  still  further,  and  possibly  an  equally  important,  point  to  bear  in 
mind,  namely,  the  economic  value  of  microbes  in  food.  In  a  short 
account  like  the  present  it  will  be  impossible  to  enter  into  hypotheses 
of  pathology,  but  we  shall  at  least  be  able  to  consider  some  of  those 
interesting  experiments  which  have  been  conducted  in  the  sphere 
of  beneficial  bacteria. 

The  injurious  effects  of  organisms  contained  in  foods  has  been 
elucidated  by  the  excellent  work  of  the  late  Dr  Ballard.  From  the 
careful  study  of  a  number  of  epidemics  due  to  food  poisoning,  he 
was  able,  without  the  aid  of  modern  bacteriology,  to  arrive  at  a 
simple  principle  which  must  not  be  forgotten.  Food  poisoning  is 
due  either  to  bacteria  themselves  or  to  their  products,  which  are 
contained  in  the  substance  of  the  food.  In  cases  of  the  first  kind, 
bacteria  gaining  entrance  to  the  human  alimentary  canal  set  up  their 
specific  changes  and  produce  their  toxins,  and  by  so  doing  in  course 
of  time  bring  about  a  diseased  condition,  with  its  consequent 
symptoms.  On  the  other  hand,  if  the  products,  sometimes  called 
ptomaines,  are  ingested  as  such,  the  symptoms  set  up  by  their  action 
in  the  body  tissues  appear  earlier.  From  these  facts  Dr  Ballard 
deduced  the  simple  principle  that  if  there  is  no  incubation  period  or, 
at  all  events,  a  comparatively  short  space  of  time  between  eating 
the  poisoned  food  and  the  advent  of  disease,  the  agents  of  the  disease 
are  products  of  bacteria,  ([f,  on  the  other  hand,  there  is  an  incuba- 
tion period,  the  agents  are  probably  bacteria.  ) 

It  is  necessary  to  mention  two  other  facts.  Dr  Cautley  has 
isolated  from  poisoned  foods  some  of  the  different  species  of  bacteria 
present.*  It  would  appear  that  these  are  limited,  as  a  rule,  to  two  or 
three  kinds.  As  regards  disease,  the  organisms  of  suppuration  are 
the  most  common.  Liquefying  or  fermentative  bacteria  are  fre- 
quently present,  the  Prnf,ML&  family  being  well  represented.  In 
addition  there  are,  according  to  circumstances,  a  number  of  common 
s.  Now,  as  we  have  pointed  out,  these 


act  injuriously  by  some  kind  of  co-operation,  or  they  may  by  them- 
selves be  harmless,  and  pathological  conditions  be  due  to  the 
occasional  introduction  of  pathogenic  species. 

The  other  fact  requiring  recognition  from  any  one  who  proposes 
to  study  the  bacteriology  of  milk,  or  indeed  of  other  foods,  is,  that  a 
not  inconsiderable  amount  of  the  evil  results  of  food  poisoning 
depends  upon  the  tj.ssues_of  the  ipdivifhml  irmAfijfrjpg  f.Ka-J'^ 

*  Report  of  Medical  Officer  to  Local  Government  Board,  1895-96,  Appendix. 


180  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

There  is  ample  evidence  in  support  of  the  fact  that  not  all  the 
persons  partaking  of  infected  milk  suffer  equally,  and  occasionally 
some  escape  altogether.  We  know  little  or  nothing  of  the  causes  of 
such  modification  in  the  effect  produced.  It  may  be  due  to  other 
organisms,  or  chemical  substances  already  in  the  alimentary  canal  of 
the  individual,  or  it  may  be  due  to  some  insusceptibility  or  resistance 
of  the  tissues.  Be  that  as  it  may,  it  is  a  matter  which  must  not  be 
neglected  in  estimating  the  effects  of  food  contaminated  with  bacteria 
or  their  products. 

There  are  few  liquids  in  general  use  which  contain  such  enor- 
mous numbers  of  germs  as  milk.  To  begin  with,  milk  is  in  every 
way,  physical  and  physiological,  admirably  adapted  to  be  a  favourable 
medium  for  bacteria.  It  is  constituted  of  all  the  chief  elements  of 
the  nutriment  upon  which  bacteria  live. 

Briefly,  we  may  summarise  the  full  diet  of  bacteria  as  :  —  nitro- 
genous matter  (proteids)  ;  non-nitrogenous  matter  containing  carbon 
and  hydrogen  (carbohydrates);  calcium,  potassium,  phosphates,  etc. 
(salts)  ;  and,  for  some  species,  oxygen.  When  we  turn  our  atten- 
tion to  milk  as  a  medium  for  bacteria,  we  find  a  complete  bacterial  diet 
—  proteids  represented  by  casein  and  lactalbiunin,  4  per  cent,  in 
total  —  carbohydrates  represented  by  lactose,  the  most  readily  affected 
of  all  the  sugars  by  bacteria;  fat  as  palmitin  and  olein;  salts, 
potassium  and  calcium  largely  as  phosphates,  the  calcium  phosphate 
being  united  with  casein.  Even  the  normal  reaction  of  milk, 
neutral  or  amphoteric,  is  favourable  to  the  growth  of  bacteria,  most 
of  which  find  a  definitely  acid  or  a  definitely  alkaline  reaction 
inimical  to  their  growth.  It  is  true  that  changes,  mostly  of  a 
fermentative  nature,  rapidly  set  in,  which  affect  milk  as  a  medium 
for  bacteria.  But  in  its  fresh,  normal,  untreated  condition  we  have 
theoretically  an  almost  ideal  medium  for  both  saprophytic  and 

ithstandi 


Notwithstanding  the  truth  of  this  general 
statement,  we  must  not  pass  over  the  experiments  of  Fokker, 
Freudenreich,  Cunningham,  and  others,  which  appear  to  demonstrate 
that  freshly-drawn  milk  possesses  for  certain  species  of_hacJteri_a  a 
germicidal  power. 

In  the  healthy  condition  of  animals  we  have,  generally  speaking, 
no  micro-organisms  whatever  in  their  secretions,  whatever  may  be  the 
condition  of  their  excretions.  Hence,  though  milk  affords,  from  its 
constitution,  such  an  ideal  nidus  for  the  growth  and  multiplication  of 
bacteria,  it  is,  as  secreted,  a  perfectly  sterile  fluid..  This  was  demon- 
strated more  than  twenty  years  ago  by  Lister,  who  states  that 
"  unboiled  milk  as  coming  from  a  healthy  cow,  really  contains  no 
material  capable  of  giving  rise  to  any  fermentative  change,  or  to  the 
development  of  any  kind  of  organism  which  we  have  the  means  of 


SOURCES  OF  POLLUTION  181 

discovering."  *  Subsequent  experiment  has  only  confirmed  the 
general  truth  of  this  statement. f  With  efficient  precautions  it  is 
possible  to  draw  from  the  udder  of  a  healthy  cow  perfectly  sterile 
milk,  which  retains  its  sterility  unchanged  for  long  periods  of  time 
in  a  sterilised  and  sealed  flask.  Yet  we  know  by  practical  experi- 
ence as  well  as  by  ultimate  changes  in  the  milk,  that,  generally 
speaking,  the  presence  therein  of  bacteria  is  very  marked. 

Sources  of  Pollution  of  Milk 

These  are  various,  and  depend  upon  many  minor  circumstances 
and  conditions.  For  all  practical  purposes  there  are  four  chief 
opportunities  between  the  cow  and  the  consumer  when  milk  may 
become  contaminated  with  bacteria: — 

1.  At  the  time  of  milking  and  during  manipulation  at  the  farm. 

2.  During  transit  to  the  town,  or  dairy,  or  consumer. 

3.  At  the  milkshop. 

4.  In  the  home  of  the  consumer. 

Pollution  at  the  time  of  milking  arises  from  the  animal,  the 
milker,  or  unclean  methods  of  milking.  It  is  now  well  known  that 
in  tuberculosis  of  the  cow  affecting  the  udder  the  milk  itself  shows 
the  presence  of  the  bacillus  of  tubercle.  In  a  precisely  similar 
manner  all  bacterial  diseases  of  the  cow  which  affect  the  milk- 
secreting  apparatus  must  inevitably  add  their  quota  of  bacteria  to 
the  milk.  To  this  matter  we  shall  have  occasion  to  refer  again. 
There  is  a  further  contamination  from  the  animal  when  it  is  kept 
unclean,  for  it  happens  that  the  unclean  coat  of  a  cow  will  more 
materially  influence  the  number  of  micro-organisms  in  the  milk  than 
the  popularly  supposed  fermenting  food  which  the  animal  may  eat. 
It  is  from  this  external  source  rather  than  from  the  diet  that 
organisms  occur  in  the  milk.  The  hairy  coat  offers  many  facilities 
for  harbouring  dust  and  dirt.  The  mud  and  filth  of  every  kind  that 
may  be  habitually  seen  on  the  hinder  quarters  of  cattle  all  contribute 
largely  to  polluted  milk.  Nor  is  this  surprising.  Such  filth  at  or 
near  the  temperature  of  the  blood  is  an  almost  perfect  environment 
for  many  of  the  putrefactive  bacteria. 

The  milker  is  also  a  source  of  risk.  His  hands,  as  well  as  the 
clothes  he  is  wearing,  can  and  do  readily  convey  both  innocent  and 
pathogenic  germs  to  the  milk.  Clothed  in  dust-laden  garments,  and 
frequently  characterised  by  dirty  hands,  the  milker  may  easily  act 
as  an  excellent  purveyor  of  germs.  Not  a  few  cases  are  also  on 
record  where  it  appears  that  milkers  have  conveyed  germs  of 

*  Transactions  of  Pathological  Society  of  London,  1878,  p.  440. 
f  See  also  Rotch,  Pediatrics,  the  Hygiene  and  Medical  Treatment  of  Children, 
London,  1896. 


182  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

disease  from  some  case  of  infectious  disease,  such  as  scarlet  fever, 
in  their  homes.  But  under  the  more  efficient  registration  of  such 
disease,  which  has  recently  characterised  many  dairy  companies, 
the  danger  of  infection  from  this  source  has  been  reduced  to  a 
minimum. 

Professor  Eussell  recounts  a  simple  experiment,  which  clearly 
demonstrates  these  simple  but  effective  sources  of  pollution :  "  A 
cow  that  had  been  pastured  in  a  meadow  was  taken  for  the 
experiment,  and  the  milking  done  out  of  doors,  to  eliminate  as  much 
as  possible  the  influence  of  the  germs  in  the  barn  air.  Without 
any  special  precaution  being  taken,  the  cow  was  partially  milked, 
and  during  the  operation  a  covered  glass  dish,  containing  a,  thin 
layer  of  sterile  gelatine,  was  exposed  for  sixty  seconds  underneath 
tliQ  belly  of  the  cow,  in  close  proximity  to  the  milk-pail.  The  udder, 
flank,  and  legs  of  the  cow  were  then  thoroughly  cleaned  with  water, 
and  all  of  the  precautions  referred  to  before  were  carried  out,  and 
the  milking  then  resumed.  A  jsecond  plate  was  then  exposed  in 
the  same  place  for  an  equal  length  of  time,  a  control  also  being 
exposed  at  the  same  time  at  a  distance  of  ten  feet  from  the  animal 
and  six  feet  from  the  ground  to  ascertain  the  germ  contents  of  the 
surrounding  air.  From  this  experiment  the  following  instructive 
data  were  gathered.  Where  the  animal  was  milked  without  any 
special  precautions  being  taken,  there  were  r325_CLbacterial  germs  per 
minute  deposited  on  an  area  equal  to  the  exposed  top  of  a  ten-inch 
milk-pail.  Where  the  cow  received  the  precautionary  treatment  as 
suggested  above,  there  were  only  115_^e^ms  per  minute  deposited 
on  the  same  area.  In  the  plate  that  was  exposed  to  the  surrounding 
air  at  some  distance  from  the  cow  there  were  65  bacteria.  This 
indicates  that  a  large  number  of  organisms  from  the  dry  coat  of 
the  animal  can  be  kept  out  of  milk  if  such  simple  precautions  as 
these  are  carried  out."  * 

The  influence  of  the  byre  air,  and  the  cleanliness  or  otherwise  of 
the  byre,  is  obviously  great  in  this  matter.  As  we  have  seen,  moist 
surfaces  retain  any  bacteria  lodged  upon  them ;  but  in  a  dry  barn, 
where  molecular  disturbance  is  the  rule  rather  than  the  exception, 
it  is  not  surprising  that  the  air  is  heavily  laden  with  microbic  life 
derived  from  dust,  dried  manure,  hay,  straw,  fodder,  etc.  Here  again 
many  improvements  have  been  made  by  sanitary  cleanliness  in 
various  well-known  dairies.  Still  there  is  much  more  to  be  done  in 
this  direction  to  ensure  that  the  drawn  milk  is  not  polluted  by  a 
microbe  -  impregnated  atmosphere.  Lastly,  it  should  not  be 
forgotten  that  during  the  straining  and  cooling  of  milk  there  are 
many  opportunities  of  contamination. 

The   risks  in  transit   differ   according   to   many   circumstances. 
*  H.  L.  Russell,  Dairy  Bacteriology,  p.  46. 


SOURCES  OF  POLLUTION  183 

Probably  the  commonest  source  of  contamination  is  in  the  use  of 
11  ten  ail_a_  an  d  milk-cans.     Any  unnecessary  delay  in  transit 


__ 

affords  increased  opportunity  for  multiplication  ;  particularly  is  this 
the  case  in  the  summer  months,  for  at  such  times  all  the  conditions 
are  favourable  to  an  enormous  increase  of  any  extraneous  germs 
which  may  have  gained  admittance  at  the  time  of  milking.  Thus 
we  have  (1)  the  milk  itself  affording  an  excellent  medium  and 
supplying  ideal  pabulum  for  bacteria  ;  (2)  a  more  or  less  lengthened 
railway  journey  or  period  of  transit  giving  ample  time  for  multi- 
plication ;  (3)  the  favourable  temperature  of  summer  heat.  We  shall 
refer  again  to  the  rate  of  multiplication  of  germs  in  milk.  It  has 
been  shown  that  milk  brought  into  large  cities,  such  as  London, 
Paris,  or  New  York,  has  been  travelling  often  for  as  long  as  two  to 
ten  hours,  often  under  conditions  favourable  to  pollution  or  at  least 
under  conditions  of  temperature  favourable  to  the  multiplication  of 
bacteria. 

Pollution  at  the  MilJcshqp.  —  Many  are  the  advantages  given  to 
bacteria  when  milk  has  reached  its  commercial  destination.  In  milk- 
shops  there  are  not  a  few  risks  to  be  added  to  the  already  imposing 
category.  Water  is  occasionally,  if  not  frequently,  added  to  milk  to 
increase  its  volume,  either  at  the  farm  or  the  milkshop.  Such  water  of 
itself  will  make  its  own  contribution  to  the  flora  of  the  milk,  unless 
indeed,  which  is  unlikely,  the  water  has  been  recently  and  thoroughly 
boiled  before  addition  to  the  milk.  Again,  it  is  impossible  to  suppose  . 
that  in  small  milkshops,  perhaps  of  a  general  nature  —  where  the 
milk  stands  for  several  hours,  pollution  is  avoidable.  From  a  hundred 
different  sources  such  milk  runs  the  risk  of  being  polluted.  The 
dust  of  the  shop  and  the  street  gain  access  to  the  pan  of  milk  on 
the  counter,  which,  commonly,  is  uncovered.  The  "  differ  "  and  the 
vendoisMiands  and  clothes  contribute  bacteria.  Flies  also  increase 
the  pollution. 

Pollution  in  the  Home.  —  Lastly,  there  is  pollution  from  dust  and 
dirt,  inorganic  and  organic,  in  the  home.  More  than  a  million  of 
the  population  of  London  live  in  tenements  of  two  rooms  or  less. 
Cooking,  eating,  sleeping,  cleaning,  and  sometimes  even  trade  employ- 
ments in  the  form  of  "  home-work,"  are  all  conducted  under  conditions 
of  overcrowding  and  lack  of  space.  Often  there  is  no  pantry  or 
larder,  and  consequently  the  days'  supply  of  milk  stands  in  a  dirty 
uncovered  vessel  in  the  midst  of  dirty  surroundings.  It  is  evident 
that  this  is  but  one  more  opportunity  for  pollution. 

Fore-Milk.  —  Before  proceeding,  a  word  must  be  said  respecting 
the  first  milk  which  flows  from  the  udder  in  the  process  of  milking, 
and  which  is  known  as  the  fQTp,-vn,illf..  This  portioi^  of  ffift  millr  i« 
ahvu^.s  rich  in  bacterial  life,  on  account  of  the  fact  that  it  lias 
remained  in  iho  milk-ducts  sinco  the  last  milking.  However  thorough 


184  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

the  manipulation,  there  will  always  be  a  residue  remaining  in  the 
ducts,  which  will,  and  does,  afford  a  suitable  nidus  and  incubator  for 
organisms.  The  latter  obtain  their  entrance  through  the  imperfectly 
closed  teat  of  the  udder,  and  pass  readily  into  the  milk-duct,  some- 
times even  reaching  the  udder  itself  and  setting  up  inflammation 
(mastitis).  Professor  Eussell  states  that  he  has  found  2800  germs 
in  the  fore-milk,  in  a  sample  of  which  the  average  was  only  330  per 
c.c.  Schultz  found  83,000  micro-organisms  per  c.c.  in  the  fore-milk, 
and  only  9000  in  the  mid-milk.  As  a  matter  of  fact,  most  of  this 
large  number  belong  to  the  lactic  acid  fermentation  grdup,  and  the 
fore-milk  rarely  contains  more"  than  two  or  three  species,  and  still 
moreT  rarely  any  disease-producing  bacteria.  Still,  bacteria  occur  in 
such  enormous  numbers  that  their  addition  to  the  ordinary  milk 
very  materially  alters  its  quality.  Bolley  and  Hall,  of  North  Dakota, 
report  sixteen  species  of  bacteria  in  the  fore-milk,  twelve  of  which 
produced  an  acid  reaction.  Dr  Veranus  Moore,  of  the  United  States 
Department  of  Agriculture,*  concludes  from  a  large  mass  of  data 
that  freshly-drawn  fore-milk  contains  a  variable  but  generally 
enormous  number  of  bacteria,  but  only  a  few  species,  the  last  milk 
containing,  as  compared  with  the  fore-milk,  very  few  micro-organisms. 
The  bacteria  which  become  localised  in  the  milk-ducts,  and  which  are 
necessarily  carried  into  the  milk,  are  for  the  greater  part  acid- 
producing  organisms,  i.e.,  they  ferment  milk-sugar,  forming  acids. 
.They  do  not  produce  gas.  Nevertheless  their  presence  renders  it 
necessary  to  "  pasteurise  "  as  soon  as  possible.  Dr  Moore  holds  that 
much  of  the  intestinal  trouble  occurring  in  infants  fed  with  ordinarily 
"pasteurised"  milk  arises  from  acids  produced  by  these  bacteria 
between  the  drawing  of  the  milk  and  the  pasteurisation.  Prof. 
MacFadyean  has  given  a  full  account  of  the  ways  in  which  milk 
becomes  pathogenic,  and  his  views  have  received  further  support  from 
Prof.  Delepine.f 

The  Number  of  Bacteria  in  Milk 

From  all  that  has  been  said  respecting  the  sources  of  pollution 
and  the  favourable  nidus  which  milk  affords  for  bacteria,  it  is  not 
surprising  that  a  very  large  number  of  germs  are  almost  always 
present  in  milk.  The  quantitative  estimation  of  milk  appears  more 
alarming  than  the  qualitative.  It  is  true  some  diseases  are  conveyed 
by  bacteria  in  milk,  but  on  the  whole  most  of  the  species  are  non- 
pathogenic.  Nor  need  the  numbers,  though  serious,  too  greatly 
alarm  us,  for,  as  we  shall  see  at  a  later  stage,  disease  is  due  to  other 
agencies  and  conditions  than  merely  the  bacteria,  which  may  be  the 

*  Bureau  of  Animal  Industry  Reports,  1895-96. 
f  Jour.  ofComp.  Path.,  1897,  vol.  x.,  pp.  150-189. 


TIME  AND  TEMPERATURE 


185 


vcra  causa.  In  addition  to  the  fact  that  the  high  numbers  have  but 
a  limited  significance,  we  must  also  remember  that  there  is  no 
uniformity  whatever  in  these  numbers.  The  conditions  which  chiefly 
control  them  are  (1)  time^and  (2)  temperature. 

The  Influence  of  "Time  and  Temperature.— We  have  already 
noticed,  when  considering  the  general  conditions  affecting  bacteria, 
how  potent  an  agent  in  their  growth  is  the  surrounding  temperature. 
Generally  speaking,  temperature  at  or  about  blood-heat  favours  bac- 
terial growth.  Freudenreich  has  drawn  up  the  following  table  which 
graphically  sets  forth  the  effect  of  temperature  upon  bacteria  in  milk: — 


3  hours. 

6  hours. 

9  hours. 

24  hours. 

59°  F. 

1  + 

2-5 

5 

163 

77°  F. 

2 

18-5 

107 

62,100 

95°  F. 

4 

1290 

3800 

5,370 

It  will  be  noticed  that  at  59°  F.  there  is  very  little  multiplication. 
That  may  be  accepted  as  a  rule.  At  77°  F.  the  multiplication,  though 
not  particularly  rapid  at  the  outset,  results  finally,  at  the  end  of 
the  twenty-four  hours,  in  the  maximum  quantity.  These  were 
probably  common  species  of  saprophytic  bacteria,  which  increase 
readily  at  a  comparatively  low  temperature.  During  the  subsequent 
hours,  after  the  twenty-four,  we  should  expect  a  temporary  decline 
rather  than  an  increase  in  62,000,  owing  to  the  keen  competition  con- 
sequent upon  the  limitation  of  the  pabulum.  From  a  consideration 
of  these  facts,  we  conclude  that  a  warm  temperature,  somewhat  below 
blood-heat,  is  most  favourable  to  multiplication  of  bacteria  in  milk ; 
that  the  common  saprophytic  organisms  multiply  the  most  rapidly ; 
that,  in  the  course  of  time,  competition  kills  off  a  large  number. 

Another  example  may  be  taken  from  Professor  Conn : — 

Number  of  Bacteria  per  cubic  centimetre  in  milk  kept  at  different 
temperatures. 


No.  at 
outset. 

In  12  hrs. 
at  50°. 

In  12  hrs. 
at  70°. 

In  50  hrs. 
at  50°. 

In  50  hrs.,  or  at 
time  of  curdling, 
at  70°. 

No.  of  hrs. 
to  curdling 
at  50°. 

No.  of  hrs. 
to  curdling 
at  70*. 

46,000 

39,000 

249,500 

1,500,000 

542,000,000 

190 

56 

47,000 
50,000 

44,800 
35,000 

360,000 

800,000 

127,500 
160,000 

792,000,000 
36  hours 
2,560,000,000 
42  hours 

289 
172 

36 
42 

186 


BACTERIA  IN  MILK  AND  MILK  PRODUCTS 


So  strongly  convinced  is  Conn  of  the  exceptional  influence  of 
temperature  on  the  increase  of  bacteria  in  milk,  and  the  subsequent 
souring,  that  he  holds  that  "  the  keeping  of  milk  is  more  a  matter  of 
temperature  than  of  cleanliness."  The  cooling  of  milk  immediately 
after  milking,  and  keeping  it  at  a  low  temperature,  will  do  more  for 
its  preservation  than  any  other  practical  device.  Conn  has  also 
pointed  out  that  lactic  organisms  flourish  in  milk  when  it  is  kept  at 
temperatures  above  50°  C.  He  summarises  the  influence  of  tempera- 
ture as  follows : — 

(1)  Variations  in  temperature  have  a  surprising  influence  upon 
the  rate  of  multiplication  of  bacteria.  At  50°  F.  these  organisms  may 
multiply  only  five-fold  in  twenty-four  hours,  while  at  70°  they  may 
multiply  seven  hundred  and  fifty-fold.  (2)  Temperature  has  a  great 
influence  upon  the  keeping  property  of  milk.  Milk  kept  at  95°  (heat 
of  the  cow's  body)  will  curdle  in  eighteen  hours,  while  the  same  milk 
kept  at  70°  will  not  curdle  for  forty-eight  hours,  and  if  kept  at  50°, 
the  temperature  of  an  ice-chest,  may  sometimes  keep  without  curdling 
for  two  weeks  or  more.  (3)  So  far  as  the  keeping  property  of  milk 
is  concerned,  the  matter  of  temperature  is  of  more  significance  than 
the  original  contamination  of  the  milk  with  bacteria.  (4)  Milk;  pre- 
<served  at  50°  or  lower  will  keep  sweet  for  a  long  time,  but  it  becomes 
filled  with,  bacteria  of  a  more  unwholesome  tvpe  than  f^na^  f.fytff  jg-nup: 
at  higher  temperatures.* 

~"  The  influence  of  time  is  not  less  marked  than  that  of  temperature, 
as  the  following  table  will  show: — 


Milk  drawn  at  59°  F. 


After 


hour 


153,000  m.o.  per  cub.  inch. 

616,000 

539,000 

680,000 

1,020,000 

2,040,000 

85,000,000 


Freudenreich  gives  another  example,  as  follows : — 


Milk  drawn  at  15 -5°  C. 
After    4    hours 
>»         9        »» 
24 


27,000  m.o.  per  c.c. 
34,000 
100,000 
4,000,000 


Concerning  these  figures  little  comment  is  necessary.  But  here 
again,  also,  we  may  remember  that  this  rapid  multiplication  only 
continues  up  to  a  certain  point,  after  which  there  is  a  marked  reduc- 
tion owing  to  products  of  activity. 

Quite  recently  further  investigations  have  been  made  in  milk 
maintained  at  a  standard  temperature  by  various  workers.  For  the 

*  Storr's  Agric.  Expt.  Sta.  Conn.  Bull  26  (H.  W.  Conn). 


TIME  AND  TEMPERATURE 


187 


sake  of  comparison  with  other  statistics,  we  may  take  two  series 
recorded  by  Park.  In  the  first  the  temperature  was  90°  F.,  a  tem- 
perature common  in  New  York  in  hot  summer  weather,  and  the 
samples  of  milk  were  of  three  degrees  of  quality,  namely,  fresh  and 
good,  fair,  and  bad.  The  result  was  as  follows : — 


Number  of  Bacteria  per  1  c.c. 

Good  Fresh  Milk. 

Fair  Milk  from 
Store. 

Bad  quality  from 
Store. 

Original  number  of  Bacteria 
After  2  hours 

„     4       „    .         .  '       , 
„     6       „    . 

,,8       „    .         .'  -. 

5,200 
8,400 
12,400 
68,500 
654,000 

92,000 
184,000 
470,000 
1,260,000 
6,800,000 

2,600,000 
4,220,000 
19,000,000 
39,000,000 
124,000,000 

The  second  series  of  Park  was  milk  taken  from  cows  in  common 
dirty  stalls,  twenty-four,  thirty-six,  and  forty-eight  hours  after 
milking.  The  milk  was  cooled  to  52°  F.,  three  hours  after  milking, 
and  maintained  at  that  temperature,  for  the  forty-eight  hours  of  the 
experiment.  The  result,  therefore,  shows  the  effect  of  time  even 
more  exactly  than  the  first  series : — 


Average  Number  of  Bacteria  per  1  c.c.  of  Milk  at  52°  F.  (six  samples). 

After  3  hours. 

After  24  hours. 

After  30  hours.* 

After  48  hours. 

30,366 

69,433 

348,883 

1,668,333 

*  The  figures  at  36  hours  were  estimated  from  the  test  of  one  sample  only. 

Even  a  cursory  examination  of  these  figures  with  those  already 
given  will  have  shown  how  intimately  the  two  influences  of  time  and 
temperature  act  and  interact  in  relation  to  the  multiplication  of 
micro-organisms  in  milk.  They  are  scarcely  separable,  and  no  hard- 
and-fast  line  can  be  drawn  by  way  of  comparison  of  these  two 
influences. 

Eeference  may  also  be  made  to  two  investigations  made,  one  by 
Park  of  New  York,  and  the  other  carried  out  by  Swithinbank  and 
the  writer. 

The  following  figures  obtained  by  Park  show  the  development 
of  bacteria  in  two  samples  of  milk  maintained  at  different  tempera- 
tures for  twenty-four,  forty-eight,  and  ninety-six  hours  respectively. 
The  first  sample  was  obtained  under  the  best  conditions  possible,  the 
second  in  the  usual  way  (the  figures  of  this  sample  are  underlined). 


188 


BACTERIA  IN  MILK  AND  MILK  PRODUCTS 


When  received,  Specimen  No.  1  contained  3000  bacteria  per  c.c.,  and 
Specimen  No.  2,  30,000  per  c.c. 


Temperature. 

Time  which  elapsed  before  making  the  test. 

24  hours. 

48  hours. 

96  hours. 

168  hours. 

32°  F.  (0°  C.)    . 

ft 
39°  F.  (4°C.)    . 

42°  F.  (5'5°C.). 
46°  F.  (6°  C.)    . 
50°  F.  (10°  C.)  . 
55°  F.  (13°  C.)  . 
60°  F.  (16°  C.)  . 
68°  F.  (20°  C.)  . 
86°  F.  (30°  C.)  . 
94°  F.  (35°  C.)  . 

2,400 
30,000 

2,500 
38,000 

2,600 
43,000 

3,100 
42,000 

2,100 
27,000 

3,600 
56,000 

3,600 
210,000 

12,000 
360,000 

1,850 
24,000 

218,000 
4,300,000 

500,000 
5,760,000 

1,480,000 
12,200,000 

1,400 
19,000 

4.200,000 
38,000,000 

11,600 
89,000 

18,800 
187,000 

180,000 
900,000 

540,000 
1,940,000 

3,400,000 
38,000,000 

28,000,000 
168,000,000 

450,000 
4,000,000 

25,000,000,000 
25,000,000,000 

1,400,000,000 
14,000,000,000 

25,000,000,000 
25,000,000,000 

There  are  two  points  in  this  table  which  may  be  noted.  First,  it 
may  be  seen  that  at  32°  F.  (0°  C.)  there  is  a  decline  in  the  number  of 
organisms  both  in  good  and  bad  milk  during  the  first  168  hours.  At 
all  the  other  temperatures,  to  which  there  is  no  exception,  there  is  a 
rise  in  the  number  of  organisms.  Secondly,  the  numbers  of  bacteria 
at  20°  C.  in  forty-eight  hours  are  equal  to  the  numbers  at  35°  C.  in 
twenty-four  hours,  and  in  both  instances  the  number  is  phenomenally 
high. 

In  1900,  Mr  Swithinbank  and  the  writer  conducted  a  series  of 
experiments  as  part  of  an  inquiry  into  the  behaviour  of  bacteria  in 
milk,  during  which  careful  observation  was  made  of  a  certain  milk 
from  the  time  it  was  drawn  from  the  udder  up  to  thirty  days,  and  then 
subsequently  after  two  years.  Further,  the  observations  were  made 
at  three  different  temperatures.  Broadly  speaking,  the  conclusions 
were  as  follow : — 

First,  there  was  an  extremely  rapid  increase  in  the  number  of 
organisms  in  the  first  four  hours,  particularly  at  37°  C.  At  the 


TIME  AND  TEMPERATURE  189 

commencement  the  milk  contained  812,000  bacteria  per  c.c.  After 
four  hours  it  contained  2,066,000  (at  5°  C.),  3,650,000  (at  15°  C.), 
and  6,116,000  (at  37°  C.). 

Secondly,  speaking  in  a  general  way,  the  following  great  principle 
became  evident,  namely,  that  there  is  at  each  temperature  (a\  a 
sudden  jrise,  (b)  a  sudden  fall,  (c)  a  steady  rise  to  maximum,  and  (d) 
a  steadj"fal_l  ultimaTely  to  sterility.'  In  other  words,  there  are  tides 
of  organisms,  and  this  was  found  to  occur  invariably  in  our  study 
of  "  natural "  milks.  It  is  a  variable  phenomenon  in  ordinary  milks, 
but  is  the  rule  in  respect  to  "  natural "  milk  examined  immediately 
after  milking.  It  is  obvious  that  if  we  had  commenced  our  examina- 
tion, as  is  frequently  the  case  in  the  study  of  town  milks,  twelve 
or  twenty  hours  after  milking,  we  should,  even  if  we  had  obtained 
the  same  figures,  have  drawn  very  different  deductions,  because  the 
initial  rise  and  initial  fall  would  have  been  lost  sight  of.  The 
sudden  fall  occurred  in  forty-eight  hours  at  5°  C.,  in  twelve  hours 
at  15°  C.  and  37°  C. 

Thirdly,  the  maximum  number  of  bacteria  occurred  in  ten  days 
at  5°  C.  (406,400,000  bacteria  per  c.c.),  in  six  days  at  15°  C. 
(84,000,000),  and  in  seventy-two  hours  at  37°  C.  (8,360,000).  The 
maximum  was  lowest  at  blood-heat  and  highest  at  5°  C.  It  is 
evident,  therefore,  that  what  occurs  in  a  short  time  at  a  high 
temperature  occurs  in  a  longer  period  at  a  low  temperature,  but  at 
a,  low  temperature  the  bacteria  eventually  become  most  numerous. 
Tliese  facts  are  of  great  importance  in  relation  to  the  time  which 
milk  is  kept  before  use,  and  to  the  injurious  properties  which  it 
may  acquire  during  such  a  period  in  the  direction  of  increased 
bacterial  toxin  production. 

Fourthly,  marked  acidity  commenced  between  the  twelfth  and 
sixteenth  hours  in  the  sample  at  37° ;  between  the  twentieth  and 
twenty-fourth  hours  at  15°;  and  between  the  seventy-second  and 
ninety-sixth  hours  at  5°  C.  At  the  end  of  these  particular  stages 
it  will  be  noticed  that  there  is  a  rising  tide  following  the  "  low-water 
mark"  of  organisms  at  each  temperature.  The  relation  which  the 
degree  of  acidity  bears  to  the  bacterial  content  is  an  intimate  one. 
As  far  back  as  1878  Lister  pointed  out  the  marked  inhibitory  effect 
which  the  presence  of  a  high  degree  of  lactic  acid  had  upon  common 
moulds  and  ordinary  saprophytic  bacteria.*  When  the  lactic  acid 
d_o<;,  lines,  these  other  forms  commence  growth,  and  eventually 
enormously  preponderate. 

Fifthly,  as  the  flasks  of  milk  were  kept  intact,  we  were  able  to 
repeat  the  experiment  in  every  particular  after  the  lapse  of  exactly 
two  years  from  the  commencement.  The  milk  was  the  same  milk, 

*  Path.  Soc.  Trans.,  1878,  p.  440. 


190  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

and  the  experiment  was  repeated  as  at  thirty  days.  During  the 
intervening  period  the  flasks  had  been  kept,  hermetically  sealed,  at 
the  three  temperatures.  The  result  was  that  the  flasks  were  found 
to  be  germ-free,  with  the  exception  of  an  abundant  growth  of  Oidmm 
lactis  and  other  moulds. 

Mr  Swithinbank  and  the  writer  came  to  the  conclusion  that  the 
explanation  of  the  results  of  this  investigation  could  only  be  found 
in  a  glance  at  the  life-history  of  such  a  milk  as  that  under 
consideration. 

"  At  the  time  of  milking  there  is,  as  we  have  seen,  an  introduction  into  the  warm 
milk  of  vast  numbers  of  common  saprophytic  and  parasitic  bacteria.  Finding 
themselves  in  an  ideal  nidus,  they  multiply  with  almost  incredible  rapidity.  Hence 
the  first  rise  in  numbers  of  bacteria.  Competition  and  exhaustion  of  pabulum 
soon  produce  inevitable  effects,  and  we  obtain  the  first  decline.  At  this  stage  it 
may  be  said  that  the  common  extraneous  bacteria,  whether  putrefactive  or  simple 
saprophytes,  practically  die  out,  and  that  for  a  very  simple  reason,  namely,  that 
they  cannot  live  in  the  presence  of  the  new  tide  of  acid-forming  bacteria.  Although 
the  lactic  acid  group  of  organisms  do  not  multiply  as  rapidly  as  ordinary  sapro- 
phytes, they  reach  a  much  higher  maximum  in  the  end.  It  is  to  this  family  of 
bacteria  that  the  second  and  maximum  r(ise  is  due.  In  time,  also,  the  same 
inimical  conditions  begin  to  act,  and  the  lactic  acid  bacteria  decline  owing  to  the 
acidity  and  to  the  lack  of  pabulum.  Eventually,  the  medium,  which  twenty  days 
before  was  an  ideal  one  for  any  organism,  and  mostly  so  for  those  which  came 
first,  and  which  ten  days  before  was  favourable  to  lactic  acid  organisms,  is  now 
favourable  to  no  bacteria  at  all.  Accordingly,  bacteria  of  all  descriptions  gradually 
die  out,  and  the  medium  is  eventually  left  in  possession  of  Oidium  lactis  and  the 
common  moulds.  That  the  destruction  of  large  quantities  of  solid  albuminous 
substances  may  occur  simply  through  bacterial  agencies  has  been  conclusively 
shown  in  the  so-called  septic  tank  method  of  sewage  disposal.  The  death  of 
bacteria  under  these  circumstances  always  follows  shortly  after  their  enormous 
multiplication,  and  how  much  is  due  to  starvation  or  how  much  to  poisoning  by  the 
products  of  their  own  activity  it  is  impossible  to  say.  It  is,  however,  clear  that  the 
decomposition  of  large  quantities  of  albuminous  substance  is  first  accompanied  by 
great  bacterial  reproduction,  and  this  is  invariably  followed  by  a  season  of  speedy 
and  extreme  mortality  of  bacteria.  In  a  general  way  that  represents,  we  believe, 
the  changes  taking  place  as  represented  in  the  record  we  have  considered.  (That 
there  are  two  rises  and  two  falls  in  the  number  of  bacteria,  the  first  rise  being  due 
to  extraneous  organisms,  and  the  second  rise  to  lactic  acid  organisms,  we  believe  to 
be  the  almost  universal  rule  in  untreated  '  natural '  milk. "  ^v 

The  effect  of  temperature  and  time  has  been  illustrated  by  Dr 
Buchanan  Young's  researches  into  the  numbers  of  bacteria  in  milk 
according  to  season,  and  the  results  of  which  were  laid  before  the  Royal 
Society  of  Edinburgh.  He  estimated  in  the  Edinburgh  milk  supply  that 
three  hours  after  milking  there  were  24,000  micro-organisms  per  c.c. 
in  winter ;  44,000  in  spring  ;  173,000  in  late  summer  and  autumn. 
Again,  he  found  that  five  hours  after  milking  there  were  41,000 
micro-organisms  per  c.c.  in  country  milk,  and  more  than  350,000 
micro-organisms  per  c.c.  in  town  milk.  Many  London  milks  would 

*  Bacteriology  of  Milk  (Swithinbank  and  Newman),  1903,  p.  135. 


TIME  AND  TEMPERATURE  191 

exceed  500,000  per  c.c.*  In  summer  the  writer  has  found  as  many  as 
4,000,000  organisms  per  e.c.  in  fresh  London  milk  obtained  at  first- 
rate  milkshops.f 

There  is  no  standard  or  uniformity  in  the  numerical  estimation 
of  bacteria  in  milk.  A  host  of  observers  have  recorded  widely- 
varying  returns  due  to  the  widely-varying  circumstances  under 
which  the  milk  has  been  collected,  removed,  stored,  and  examined, 
and  due  also  to  the  two  dominating  influences  of  time  and  tempera- 
ture. Nor  is  it  possible  to  establish  any  standard  which  may  be 
accepted  as  a  normal  or  healthy  number  of  bacteria,  as  is  done  in 
water  examination.  Bitter  has  suggested  50,000  micro-organisms 
per  c.c.  as  a  maximum  limit  for  milk  intended  for  human  consump- 
tion, but  actual  experience  shows  that,  at  present  at  all  events,  such 
standards  are  impracticable. 

Owing  to  differences  of  nomenclature  and  classification,  in  addition 
to  differences  in  mode  of  examination,  at  present  existing  in  various 
countries,  it  is  impossible  to  state  even  approximately  how  many 
bacteria,  and  how  many  species  of  bacteria,  have  been  isolated  from 
milk.  Until  some  common  international  standard  is  established, 
mathematical  computations  are  practically  worthless.  They  are  need- 
lessly alarming  and  sensational.  And  it  should  be  remembered  that 
great  reliance  cannot  be  placed  upon  these  numerical  estimations,  for 
they  vary  from  day  to  day,  and  even  hour  to  hour.  Furthermore,  vast 
numbers  of  bacteria  are  economic  in  the  best  sense  of  the  term,  and 
the  bacteria  of  milk  are  chiefly  those  of  a  fermentative  kind,  and 
notdisease^producers.  J 

The  effects  of  time  and  temperature  upon  the  bacteria  of  milk  do 
not  only  concern  the  numbers  of  organisms  present.  As  long  ago  as 
1897  Delepine  showed  that  fgwrify  nf  milk  W^Jn^T^asH  by  a  rise 
in  temperature,  and  this  is  as  we  should  expectT^or  it  stands  to 
reason  f,|ipt  nrmrlitiffng  4W™i™^|fl  fn  the  multiplication  of  bacteria  in 
milk  must  of  necessity  tend  to  increase  the  products  of  bacteria  in 
milk,  and  is  likely  to  increase  its  virulence.§ 

The  following  tallies  summarise  the  more  detailed  figures  given 
in  1897,  and  in  which  the  effects  which  length  of  keeping  and 
of  temperature  have  upon  the  noxious  effects  of  the  milk  are 
indicated. 

*  Brit.  Med.  Jour.,  1895,  ii.,  p.  322. 

t  Report  on  Milk  Supply  of  Finsbury,  1903. 

£  At  the  same  time  it  is  important  to  remember  that  comparative  series  of 
estimations  as  to  the  number  of  bacteria  per  c.c.  in  milk  may  be  of  value  as 
indication  of  unclean  dairying.  Leighton  of  Montclair,  U.S.A.,  has  shown  that 
numbers  increase  in  direct  proportion  to  unclean  management.  See  The  Milk 
Supply  of  Two  Hundred  Cities  and  Towns  (Alvord  &  Pearson),  U.S.  Dep.  of 
Agriculture,  Bull.  46,  1903,  p.  117. 

§  Jour.  ofComp.  Path,  and  Therapeutics,  1897. 


192 


BACTERIA  IN  MILK  AND  MILK  PRODUCTS 


Delepine  showed  that  mixed  milks  coming  from  a  distance  of  over 
40  miles,  and  generally  kept  for  from  twenty-four  to  sixty  hours,  and 
even  more  in  a  few  cases  (tuberculous  samples  excluded),  gave  the 
following  returns : — 


Mean  Temperature  in  the 
Shade  (Manchester) 
during  Time  the  Specimens 
were  kept. 

Specimens 
producing 
no  Noxious 
Effects. 

Noxious 
Specimens. 

Totals. 

Percentage 
of  Good 
Specimens. 

Deg.  Fahr. 

30  to  35 

7 

5 

12 

58'0 

35  to  40 

7 

11 

18 

38-5 

40  to  45 

2 

3 

5 

40-0 

45  to  50 

1 

4 

5 

20-0 

50  to  55 

55  to  60 

0 

2 

2 

o-o 

17 

25 

42 

39-0 

Mixed  milks  coming  from  a  short  distance  (generally  under  20 
miles),  most  of  them  kept  for  less  than  ten  hours  (with  the  excep- 
tion of  five  out  of  the  seven  bad  specimens,  and  four  out  of  the 
twenty-two  good  specimens,  which  had  been  kept  somewhat  longer), 
(tuberculous  samples  excluded),  gave  the  following  results  : — 


Mean  Temperature  in  the 
Shade  (Manchester) 
during  Time  the  Specimens 
were  kept. 

Specimens 
producing 
no  Noxious 
Effects. 

Noxious 
Specimens. 

Totals. 

Percentage 
of  Good 
Specimens. 

Deg.  Fahr. 
50  to  55 

1 

0 

1 

lOO'O 

55  to  60 

8 

1 

9 

88-8 

60  to  65 

11 

4 

15 

73-2 

65  to  70 

... 

... 

70  to  75 

2 

2 

4 

50'0 

22 

7 

29 

75-68 

Whilst  unmixed,  milks  kept  for  various  lengths  of  time,  but 
collected  from  the  udder  in  sterilised  vessels  (tuberculous  samples 
excluded),  resulted  as  follows  : — 


TIME  AND  TEMPERATURE 


193 


Mean  Temperature  in  the 
Shade  (Manchester) 
during  Time  the  Specimens 
were  kept. 

Specimens 
producing 
no  Noxious 
Effects. 

Noxious 
Specimens. 

Totals. 

Percentage 
of  Good 
Specimens. 

Deg.  Fahr. 

35  to  40 

6 

0 

6 

100-0 

40  to  45 

3 

2 

5 

60-0 

45  to  50 

5 

2 

7 

71-5 

50  to  55 

55  to  60 

60  to  65 

0 

3 

3 

o-o 

14 

7 

21 

67-2 

"  The  influence  of  time,"  Prof.  Delepine  adds,  "  is  well  shown  by 
the  number  of  specimens  remaining  good,  even  at  a  high  temperature, 
when  the  milk  had  been  kept  only  half  a  day.  On  the  other  hand, 
the  influence  of  temperature  is  still  more,  evident,  for  in  every 
category  the  number  of  good  specimens  is  almost  inversely  propor- 
tional to  the  height  of  the  temperature.  Still,  it  is  important  to  keep 
the  two  factors  of  time  and  temperature  in  mind.  (What  is  produced 
in  a  few  hours  in  summer  may  also  occur  in  winter,  when  the  milk  has 
been  kept  a  long  time"  )  j 

The  converse  is  also  true,  namely,  that  fif  the  temperature  of  milk  , 
be  reduced  by  refrigeration,  the  toxicity  of   the   milk  is   lesseneo^/ 
Professor  Delepine  has  shown  that  the  mortality  from  all  causes  in 
guinea-pigs  inoculated  with  refrigerated   milk  is   considerably  less 
than  it  is  if  unrefrigerated  milk  be  inoculated : — 


Unrefrigerated  Milk  Examined  during  the  years  1896  and  1897. 

Number 
of 
Samples. 

Number    of     Samples 
causing    the    Death 
of  Two  Animals  In- 
oculated in  less  than 
Ten  days. 

Number    of    Samples 
causing    the    Death 
of   One  of  the    In- 
oculated Animals  in 
less  than  Three  Days. 

Total. 

1896-97 

148 

Per  cent. 
5     .         .3-3 

Per  cent. 
11   .          .      7'4 

Per  cent. 

10-7 

Refrigerated  Milk  Examined  from  1898  to  1901. 

1898 
1899 
1900 
1901 

111 
175 
802 
694 

0     .         .0 
1     .        .     0-57 
4    .         .     0-50 
1     .         .     0'14 

3     .          .27 
1     .  •      .     0-57 
25     .         .     3-1 
8     .         .1-1 

27 
1-14 
3-60 
1-24 

N 


194  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

"  The  inference  to  be  drawn  from  these  gross  results  is  clear :  a 
certain  proportion  of  the  samples  of  milk  contained  bacteria  which, 
under  favourable  circumstances,  gave  to  the  milk  noxious  properties, 
the  development  of  which  could  be  checked  in  many  cases  by  pre- 
venting the  growth  of  these  bacteria.  ^The  difference  between 
refrigerated  and  non-refrigerated  milk  would  have  been  very  much 
greater,  if  the  milk  had  invariably  been  cooled  immediately  after  the 
milking  of  the  cows/  (Delepine). 

Therefore,  it  may  be  said  that  to  refrigerate  milk  immediately 
after  drawing  it  from  the  cow  is  to  reduce  the  number  of  bacteria  and 
to  diminish  the  potential  toxicity  of  the  milk.  Finally,  Professor 
Delepine  writes : — 

"  When  the  clear  relation  existing  between  time  of  keeping,  plus 
temperature  and  the  noxious  properties  of  a  certain  number  of 
samples  of  milk,  is  contrasted  with  the  ambiguous  results  obtained 
when  an  attempt  is  made  to  connect  these  noxious  properties  with 
disease  of  the  udder  (tuberculosis  being  excluded),  it  is  difficult  not 
to  feel  convinced  that  infection  of  the  milk  outside  the  udder,  and 
the  conditions  under  which  milk  is  kept,  are  the  most  important 
factors  causing  it  to  acquire  infective  properties."* 

Species  of  Bacteria  found  in  Milk 

The  kinds  of  bacteria  occurring  in  milk  may  for  purposes  of  con- 
venience be  classified  in  the  following  four  divisions  ;  though  of  the 
first  two  groups  it  is  not  necessary, to  say  much  here : — 
•^  1.  Ordinary  bacteria  of  air,  soil,  or  water. 
-  2.  Bacteria  of  sewage  or  intestinal  origin. 

3.  Bacteria  concerned  in  fermentation. 

4.  Pathogenic  bacteria,  in  particular  those  associated  with  tuber- 
culosis, enteric  fever,  cholera,  scarlet  fever,  diphtheria,  sore-throat 
illnesses,  and  epidemic  diarrhoea. 

1.  Ordinary  bacteria  of  soil,  air,  or  water  readily  gain  access  to 
milk  from  their  natural  media.     It  is  unnecessary  to  consider  them 
here. 

2.  Bacteria  of  sewage  and  intestinal  origin  occur  from  time  to  time 
in  milk.     The  two  chief  representatives  are  B.  coli  and  B.  enteritidis 
sporogenes.     In  Liverpool,  from  1900-1902,  788  "  country  "  milks  were 
examined,  and  55  per  cent,  contained  B.  coli  and  9  per  cent,  contained 
B.  enteritidis  sporogenes ;  of  722  "  town  "  milks,  23  per  cent,  contained 
the  former  bacillus,  and  4  per  cent,  the  latter.-f     Chick  found  B.  coli 
present  in  17  out  of  239  new  milks,  and  Balfour  Stewart  found  B. 
enteritidis  sporogenes  in  49  samples  out  of  213.     When  it  is  considered 

*  Jour,  of  Hygiene,  1903,  pp.  80-84. 
f  Bacteriology  of  Milk,  p.  216. 


COMPOSITION  OF  MILK  195 

how  filthily  many  cows  are  kept,  it  is  not  to  be  wondered  that  many 
intestinal  organisms  find  their  way  to  milk. 

3.  Bacteria  concerned  in  fermentations  in  milk  cannot  well  be 
understood  without  some  appreciation  of  the  different  elements  of 
milk  which  are  most  affected  by  the  changes  of  fermentation.  It  is 
therefore  necessary,  before  proceeding,  to  consider  shortly  what  are 
the  constituents  of  milk  upon  which  living  ferments  of  various  kinds 
exert  their  action,  for  without  these  facts  the  action  of  fermentation 
bacteria  is  not  evident.  A  tabulation  of  the  chief  constituents  of 
milk  may  be  stated  as  follows  : — 


(1)  Water 

Ordinary  (2)  Milk-sugar 

fresh  milk  =  \      (3)  Fat 

100  per  cent.   |      (4)  Proteids  (casein,  etc.) 

(5)  Mineral  matter      . 


8 7 '5  per  cent. 
4'9 
3-6 
3'3 
0-7 


lOO'O 


Or  the  average  milk  constitution  may  be  expressed  thus  : — 

Fat  ......         4-1  per  cent. 

Solids  not  fat        .  .  .  .  .         8*8        „ 

Total  solids          .  .  .  .       12-9        ,, 

Water       ......       S7'l 

It  is  not  necessary  to  remark  that  milks  vary  in  standard,  and  the  above  figures 
can  only  be  taken  as  fair  averages. 

Milk-sugar,  or  Lactose  (C12H24O12),  is  an  important  and  constant  constituent  of 
milk.  It  forms  the  chief  substance  in  solution  in  whey  or  serum,  and  is  a  member 
of  the  cane-sugar  group.  Milk-sugar  is  found  in  varying  quantities  in  the  milk  of 
mammals.  About  5  per  cent,  is  present  in  human  milk,  and  somewhat  less  in  that 
of  the  cow.  It  is  very  resistant  to  fermentation  by  yeast,  and  therefore  undergoes 
alcoholic  fermentation  very  slowly.  It  is  not  acted  upon  by  rennet,  pepsin,  or 
trypsin.  But  of  all  the  sugars  it  is  most  readily  acted  upon  by  micro-organisms. 

Fat  occurs  in  milk  as  suspended  globules  of  varying  size.  It  forms  the  cream, 
and  by  churning  is,  of  course,  made  into  butter,  though  both  cream  and  butter  con- 
tain other  constituents  besides  fat.  Lloyd  has  shown  that  it  is  the  large  globules 
that  form  the  cream,  and  he  has  also  made  observations-upon  the  size  of  fat  globules 
in  relation  to  breed  of  cattle.  The  decomposition  and  breaking  down  of  milk-fat  by 
fermentation  is  the  chief  cause  of  gross  abnormalities  of  cream  and  the  rancidity  of 
butter. 

The  Proteids  of  Milk  include  casein,  lactalbumin,  and  lactoglobulin.  Casein  is 
by  far  the  most  abundant  and  the  most  important.  When  milk  separates  naturally 
into  its  constituent  parts  the  fat  rises  and  the  casein  falls,  leaving  a  clear  fluid,  the 
milk  plasma  or  serum,  between  the  two  substances.  The  changes  set  up  in  casein 
by  bacteria  are  various,  and  furnish  a  means  of  diagnosis. 

Mineral  Matter. — The  ash  of  milk,  obtained  by  careful  ignition  of  the  solids, 
contains  calcium,  magnesium,  potassium,  sodium,  phosphoric  acid,  sulphuric 
acid,  chlorine,  and  iron — phosphoric  acid  and  lime  being  present  in  the  largest 
amounts. 

We  may  now  consider  the  fermentations  of  milk  and  the  pathogenic 
organisms  associated  with  milk. 


196  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 


1.  THE  FERMENTATIONS  OF  MILK 

(1)  Lactic  Acid  Fermentation:  the  Souring  of  Milk.—  It  milk  is 
loft  undisturbed,  it  is  well  known  that  eventually  it  becomes  sour. 
^he  casein  is^  coagulated,  and  falls  to  the  bottom  of  the  vessel  ;  the 
whey  or  scrum  rises,  carrying  to  the  surface  flakes  or  lumps  of  fat. 
In  fact,  a  coagulation  analogous  to  the  clotting  of  blood  has  taken 
place.  In  addition  to  this,  the  whole  has  acquired  an  acid  taste. 
Now  this  double  change  is  not  due  to  any  one  of  the  constituents  we 
have  named  above.  It  is,  in  short,  a  fermentation  set  up  by  a  living 
ferment  introduced  from  without.  The^  constituents  Hflfo  ftffftftte^ 
by  the  fermentation  are  (a)  the  milk-sugar,  which  is  broken._dawn 
into  lactic  acid,  carbonic  acid  gas,  arid  other  products,  and  (&).  .the 
casein,  which  is  curdled  and  becomes  suspended  in  a  semi-colloidal 
form. 

For  many  years  it  has  been  known  that  sour  milk  contained 
bacteria.  Pasteur  first  described  the  Bacillus  acidi  lactici,  which 
Lister  isolated  in  1877,  and  obtained  in  pure  culture  by  the  dilution 
method.  In  1884  Hueppe  contributed  still  further  to  what  was 
known  of  this  bacillus,  and  pointed  out  that  there  were  a  large 
number  of  varieties,  rather  than  one  species,  to  be  included  under 
the  term  B.  acidi  lactici.  We  have  already  dealt  with  the  chief 
characters  of  this  family  of  organisms.  When,  ascertain  quantity  of 
lactic  acid  has  been  formed,  the  fermentation  ceases.  It  will 
recommence  if  the  liquid  be  neutralised  with  carbonate  of  ..limey  or 
if  pepsine  be  added.  Since  Pasteur's  discovery  of  a  causal  bacillus 
for  this  fermentation,  other  investigators  have  added  a  number  of 
bacteria  to  the  lactic  acid  family.  Some  of  these  in  pure  culture 
have  been  used  in  dairy  industry,  as  we  shall  subsequently  have 
occasion  to  notice. 

We  have  already  seen  that  milk  as  it  leaves  the  healthy  udder 
is  generally  sterile,  and  immediately  gains  bacteria  from  air,  dust, 
etc.  Whilst  the  exact  origin  of  lactia  acid  bacilli  is  not  known, 
many  bacteriologists  hold  that  they  gain  entrance  to  the  milk  from 
tKT  surrounding  air  of  byre  or  dairy.  Others  maintain  that  some 
species,  at  any  rate,  are  soil  bacteria,  and  associated  with  certain 
geographical  localities.  Eussell  states  that,  under  ordinary  con- 
ditions, the  organisms  found  in  the  teat  of  the  udder  are  those  which 
produce  lactic  fermentation.  He  quotes  Bolley  and  Hall  as  finding 
twelve  out  of  sixteen  species  in  the  teat  of  the  udder  to  be  lactic  acid 
producers.*  Veranus  Moore  has  arrived  at  very  similar  results.f 
Rollin  Burr  has  recently  investigated  this  subject  with  a  different 

*  Outlines  of  Dairy  Bacteriology,  H.  L.  Russell,  1898,  p.  43. 
t  Twelfth  and  Thirteenth  Reports  of  the  Bureau  of  Animallndusiry,  U.S.A.,  1895 
and  1896,  p.  265. 


LACTIC  ACID  FERMENTATION  197 

result.*  He  finds  that  when  milk  is  drawn  from  the  cow  in  such  a 
manner  as  to  exclude  from  it  dirt  and  dust  from  the  air,  the  stalls,  and 
the  cow,  such  milk  may  contain  none  of  the  organisms  capable  of 
producing  a  normal  souring  of  milk.  This  also  has  been  the 
experience  of  the  writer.  Tlie  lactic  acid  organisms  are  a  secondary 
contamination  of  the  milk  from  some  external  source.  None  of  the 
species  of  lactic  organisms  characteristic  of  the  locality  in  which 
Burr  worked,  could  be  found  in  the  udder.  This  is  in  accordance 
with  the  results  of  others  who  have  had  the  opportunity  of  examining 
the  udder  or  milk  ducts  for  lactic  bacilli.  Out  of  300  examinations 
made  of  fore-milks  drawn  directly  from  the  udder  into  sterile  flasks, 
Burr  found  only  2  per  cent,  contained  ordinary  lactic  acid  bacteria, 
and  in  these  cases  the  origin  was  probably  outside  contamination. 
Conn  found  the  acid  organisms  present  in  5  cases  out  of  200 
examinations,  involving  75  cows.  He  also  maintains  that  the  origin 
of  lactic  acid  bacteria  is  in  external  conditions.-)-  Further,  there  is 
the  recognised  fact  which  has  been  pointed  out  by  Conn  and  Esten, 
and  frequently  met  with  by  Swithinbank  and  the  writer,  namely, 
that  lactic  acid  organisms  are  not  the  predominant  species  in  freshly- 
drawn  milk,  as  they  undoubtedly  would  be  were  they  organisms 
of  the  udder.  Hence  there  can,  we  think,  be  little  doubt  that  the 
origin  of  lactic  acid  organisms  is  to  be  found  in  some  external 
condition  or  conditions. 

It  follows  from  what  has  been  said  that  deanline%s  of  byrer  dairy, 
and  general  manipulation  is  an  important  factor  in  the  presence, 
both  actual  and  in  degree,  of  lactic  acid  organisms. 

(2)  Butyric  Acid  Fermentation. — This   form   of  fermentation  is 
also  one  which  we  have  previously  considered.     Both  in  lactic  and 
butyric  fermentation  we  must  recognise  that  in  the  decomposition  of 
milk-sugar   there   are  almost  always  a  number  of  minor  products 
occurring.     Some  of  the  chief  of  these  are  gases.    Hydrogen,  carbonic 
acid,  nitrogen,  and  methane  occur,  and  cause  a  characteristic  effect 
which  is  frequently  deleterious  to  the  flavour  of  the  inilk  and  its 
products.     Most  of  the  gas-producing  ferments  are  members  of  the 
lactic  acid  group,  and  are  sometimes  classified  in  a  group  by  them- 
selves.    In  butyric  fermentation  of  milk  the  three  chief  products 
are  butyric  acid  (which  causes  the  bitterness),  hydrogen,  and  carbonic 
acid  gas. 

(3)  Coagulation  Fermentations  without  Acid  Production. — Of  these 
there   are   several,  caused  by  different  bacteria.     "What  happens  is 
that  the  milk  coagulates,  but  no  acid  is  produced,  the  whey  being 
sweet  to  the  taste  rather  than  otherwise.     The  condition  is  in  the 

*  Storr's  Agricultural  Expt.  Sta.  Rep.  for  1900,  pp.  66-81.     Centralb.  f.  Bakt., 
Abth.  ii.,  1902,  p.  236. 

t  Storr's  Agricultural  Expt.  Sta.  Rep.,  1899,  p.  23, 


198  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

main  one  of  milk-clotting  rather  than  milk-curdling.  The  two  chief 
examples  are  the  rennet  fermentation  of  milk  and  the  production  of 
casease. 

(4)  The  Alcoholic  Fermentations  qf  Mttb. — Lactose  is  not  readily 
acted  upon  by  yeasts  though  they  have  the  power  of  breaking  it  up 
and  producing^alcxiliol  and  carbonic  acid  gas.  When  it  does  occur  the 
percentage  of  alcohol  is  very  small.  The  first  change  is  the  inversion 
of  the  milk-sugar  into  dextrose  and  galactose,  and  the  second  is 
fermentation  of  these  sugars. 

Occasionally,  alcohol  is  present  in  the  milk  of  a  dairy,  as  a  sort  of 
by-product  accompanying  lactic  fermentation,  and  alcoholic  fermenta- 
tion may,  under  exceptional  circumstances,  cause  serious  trouble  to 
the  dairyman.  But  the  chief  illustrations  of  this  fermentation  in 
milk  are  the  well-known  examples  of  the  artificial  beverages  known 
as  koumiss^QY  kumiss,  kumys)  and  kephir  (or  kefyr,  kefr),  the  former 
a  fermentation  of  mare's  milk,  the  latter  of  cow's  milk.  Matzoon 
and  Leben  are  two  other  examples  of  similar  changes. 

Koumiss  is  made  on  the  Steppes  of  South-Western  Siberia  and 
European  Eussia,  by  nomadic  Tartars.  It  is  not  a  simple  process 
nor  a  single  fermentation.  There  is  first  a  lactic  fermentation 
producing  lactic  acid,  and  secondly,  a  vinous  fermentation  reaujt- 
ing  in  alcohol.  The  former  is  produced  by  bacteria,  the  latter  by 
yeasts.  In  neither  case  is  the  process  set  up  by  a  pure  culture. 
"The  net  change  which  has  taken  place  in  the  original  milk  may 
be  summed  up  by  saying  that  the  sugar  has  been  to  a  large  extent 
replaced  by  lactic  acid,  alcohol,  and  carbonic  acid  gas;  the  casein  has 
been  partly  precipitated  in  a  state  of  very  fine  division,  and  partly 
predigested  and  dissolved,  while  the  fat  and  salts  have  been  left 
much  as  they  were."  *  The  total  proteid  in  koumiss  is  hardly  less 
than  in  mare's  and  cow's  milk ;  the  fat  is  practically  the  same  as  in 
mare's  milk,  and  the  sugar  is  reduced  from  about  5  per  cent,  to  1*5 
per  cent.  The  amount  of  alcohol  in  koumiss  is  as  little  as  17  per 
cent.,  and  there  is  not  as  much  as  1  per  cent,  of  lactic  acid. 

Kephir,  the  second  example  named,  is  an  effervescent  alcoholic 
sour  milk  prepared  by  inhabitants  of  the  Caucasus  from  the  milk  of 
goats,  sheep,  and  cows.  The  process  of  fermentation  is  a  double 
one,  and  precisely  parallel  to  that  occurring  in  the  production  of 
koumiss.  Its  method  of  manufacture  is  simply  to  add  to  milk  a  few 
"kephir  grains,"  allow  the  milk  to  stand  for  twenty -four  hours  at  a 
temperature  of  17°  to  19°  C.,  pour  off  the  milk  and  mix  with  fresh 
volumes,  and  so  on.  Fermentation  is  complete  in  two  or  three  days' 
time,  and  the  resultant  fluid  contains  about  2  per  cent,  of  alcohol, 
being  slightly  more  than  in  koumiss. 

*  Food  and  the  Principles  of  Dietetics,  by  R.  Hutchison,  M.D.,  F.R.C.P.,  1902, 
p.  13.6. 


ANOMALOUS  FERMENTATIONS  199 

(5)  A^wmalous_Jj^rin^ntatwns  of  Milk- — There  are  a  number  of 
changes,  mostly  due  to  fermentation,  which  occur  in  milk,  and  to 
which  reference  must  be  made.  These  conditions  have  been  termed 
"  diseases  "  of  milk,  but  it  is  not  altogether  a  satisfactory  term. 

(a)  TtjMwr_  Ferm.r.'n.f.a.t'i.nn,l — Some  bitter  conditions  of  milk  are  due 
to  irregularity  of  diet  in  the  cow.     Similar  changes  occur  in  con- 
junction with  some  of  the  acid  fermentations  and  proteid  decomposi- 
tions.    Weigmann  and  Conn  have,  however,  shown  that  there  is  a 
specific  bitterness  in  milk  due  to  bacteria,  which  appear  to  produce 
no   other  change.     Hueppe   suggests  that  it  may  be   due  in  part 
to    a   proteid   decomposition    resulting  in  J3itter_jpeptones.      Such 
bodies   are   produced   by  bacteria   from   the   alFumrnoicTs  of   milk, 
and  hence  the  bitterness  does  not  appear  immediately  after  milk- 
ing, but  only  after  an  incubation  period.      Some  nine  or  ten  different 
micro-organisms   have   been   credited   with    this   power,   and   such 
organisms  may  infect  a  farm,  a  byre,  or  a  dairy,  for  months  or  even 
years,  contaminating  the  milk.     In  all  probability,  most  outbreaks 
of  this  bitter  fermentation  are  due  to  Weigmann's  bacillus  of  bitter 
milk  or  Conn's  micrococcus.     There  seems  to  be  evidence  for  sup- 
posing that   some  of   the   "bitter"    bacilli   produce  very  resistant 
spores,  which  make  them  resistant  against  conditions  in  the  milk 
itself  or  externally. 

(b)  Slimy  fermentation. — This    graphic   but   inelegant   term   is 
used  to  denote  an  increased  viscosity  in  milk,  and  its  tendency  when 
1  KM \\g  poured  to  become  ropy  and  fall  in  strings.     Such  a  condition 
deprives  the  milk  of  its  use  in  the  making  of  certain  cheeses,  whilst 
in  other  cases  it  favours  the  process.     In  Holland,  for  example,  in 
the   manufacture   of   Edam  cheese,   this   "slimy"   fermentation   is 
desired.     TcettemcelJc,  a  popular  beverage  in  Norway,  is  made  from 
milk  that  has  been  infected  with  the  leaves  of  the  common  butter- 
wort,  Pinguicula  vulgaris,  from  which -Weigmann  separated  a  bacillus 
possessing  the  power  of  setting  up  slimy  fermentation.     There  are, 
perhaps,  as  many  as  a  dozen   species  of  bacteria  which  have  in  a 
greater  or  less  degree  the  power  of  setting  up  this  kind  of  fermenta- 
tion.    In  1882,  Schmidt-Muhlheim  isolated  the  Micrococcus  viscosus, 
which  occurs  in  chains  and  rosaries,  affecting  the  milk-sugar.      It 
grows  at  blood-heat,  and  is  not  easily  destroyed  by  cold.     Its  effect 
on  various  sugars  is  the  same.     M.  Freudenreichii,  one  of  the  specific 
micro-organisms  of  "  ropiness  "  in  milk,  is  a  large,  non-motile,  lique- 
fying coccus,  which  can  produce  its  result  in  milk  within  five  hours. 
On  account  of  its  resistance  to  drying,  it  is  difficult  to  eradicate 
when  once  it  makes  its  appearance  in  a  dairy.     The  organism  used 
in  making  Edam  cheese  is  the  Streptococcus  Hollandicus,  and  in  hot 
milk  it  can  produce  ropiness  in  one  day.     A  number  of  bacilli  have 
been  detected  by  several  observers,  and  classified  as  slime  fermenta- 


200  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

tion  bacteria.  The  Bacillus  lactis  pituitosi,  a  slightly  curved,  non- 
liquefying  rod,  which  is  said  to  produce  a  characteristic  odour,  in 
addition  to  causing  ropiness,  brings  about  some  acidity.  B.  lactis 
mscosus  of  Adametz,  B.  actinobacter  of  Duclaux,  B.  Hessii  of  Guille- 
beau,  and  other  bacilli  are  similar  agents.  Many  of  the  above 
organisms,  with  others,  produce  "  slimy "  fermentation  in  alcoholic 
beverages  as  well  as  in  milk. 

(c)  So&py--llfttk~is  another  form  of  fermentation,  the  etiology  of 
which    has    been    elucidated    by   Weigmann.      The   Bacillus   lactis 
saponacei  imparts  to  milk  a  peculiar  soapy  flavour.     Ij^was  de.frectgd^ 
in  the  straw  of  the  bedding  and  hay  of  the  fodder,  and  from  such 
sources   may  infect  the  milk.     There  is   little   or   no  coagulation, 
but  a  certain  amount  of  sliminess  and   ropiness,  with  a  peculiar 
soapy  taste  to  the  milk. 

(d)  Chromogenie    Changes^ — We    have    already  remarked   that 
colour  is  the^natural  and  apparently  chief  product  of  many  of  the 
innocent  bacteria.     They  put  out  their  strength,  so  to  speak,  in  the 
production  of  bright  colours.     The  chief  colours  produced  by  germs 
in  milk  are  as  follows : — 

Red  Milk. — Bacillus  prodigiosus,  in  the  presence  of  oxygen,  causes 
a  redness,  particularly  on  the  surface  of  milk.  It  was  possibly  the 
work  of  this  bacillus  that  caused  "  the  bleeding  host "  which  was  one 
of  the  superstitions  of  the  Middle  Ages.  B>  lactis  erythrogenes  pro- 
duces a  red  colour  only  in  the  dark,  and  in  milk  that  is  nut  strongly 
acid  in  reaction.  When  grown  in  the  light  this  organism  produces  a 
yellow  colour.  There  is  a  red  sarcina  (Sarcina  rosca)  which  also 
has  the  faculty  of  producing  red  pigment.  One  of  the  yeasts  is 
another  example.  It  must  not  be  forgotten  that  redness  in  milk 
may  actually  be  due  to  the  presence  of  blood  from  the  udder  of  the 
cow. 

It  is  of  importance  clearly  to  differentiate  between  milk  reddened 
by  the  admixture  of  blood  from  the  mammary  gland,  and  that  pro- 
duced by  the  organism  isolated  and  studied  by  Hueppe  and  Groten- 
feldt — Bacillus  lactis  erythrogenes — the  presence  of  which  in  the 
milk  is  now  looked  upon  as  the  active  causation  of  the  disease.  In 
the  former  case  the  coloration  is  apparent  immediately  after  milking, 
is  uniform,  and  if  the  milk  is  allowed  to  rest  the  flocculent  blood 
coagulum  causing  the  coloration  will  gradually  sink  and  deposit 
itself  in  the  form  of  a  precipitate  at  the  bottom  of  the  milk 
receptacle.  In  the  latter  the  red  spots  do  not  appear  until  later,  the 
infection  of  the  milk  is  comparatively  slow,  and  the  milk  serum  is 
alone  affected,  the  cream  layer  not  taking  the  red  coloration. 
This  is  probably  due  to  the  simple  fact  that  the  cream  layer  being 
at  the  surface  is  exposed  to  the  light,  which  inhibits  the  coloration. 
A  general  coagulation  of  the  milk  takes  place  accompanied  by  a 


ANOMALOUS  FERMENTATIONS  201 

distinctive  sickly  sweetish  odour.  The  red  coloration  will  not 
occur  if  the  milk  is  exposed  to  light  or  has  an  acid  reaction.  The 
Bacillus  lactis  erythrogenes  of  Hueppe  (Bacterium  erythrogenes  of 
Grotenfeldt)  is  an  aerobic,  liquefying,  non-motile,  non-sporing, 
chromogenic  bacillus  of  1  to  1*5  mm.  in  length  by  *3  to  -4  mm.  in 
breadth,  at  times  attaining,  especially  in  broth  cultures,  a  length  of 
4  to  5  mm.  in  the  form  of  filaments.  It  takes  readily  all  ordinary 
stains  and  holds  the  Gram.  In  sterile  milk  a  gradual  precipitation 
of  the  casein  takes  place  with  a  neutral  or  slightly  alkaline  reaction 
of  the  medium.  The  resultant  serum,  in  the  absence  of  light,  absorbs 
the  red  colouring  matter  produced  by  the  organisms,  taking  a  deep 
red  tint  provided  the  medium  has  no  acid  reaction.  The  coagulation 
by  rennet  of  milk  infected  with  the  organism  has  the  effect  of 
producing  a  marked  dirty  red  coloration,  changing  to  a  reddish- 
brown  and  finally  to  blood  red. 

Blue  Milk,  is  due  to  the  growth  of  the  Bacillus  cyanogencs 
(Bacterium  syneyaneum  of  Ehrenberg),  or  as  Hueppe  originally 
termed  it,  the  Bacillus  lactis  cyanogenus,  an  anaerobic,  non-liquefying 
bacillus,  motile,  bi-polar,  flagellated,  chromogenic,  and  round-ended, 
with  a  varying  average  length  of  from  1  to  4  mm.  by  '3  to  '5  mm.  in 
breadth.  Spore  _  formation  has  been  claimed  by  Hueppe,  but 
denied  by  Heim,  who  describes  the  so-called  spores  of  Hueppe  as 
involution  forms  only.  In  liquid  cultures  curious  involution  forms 
are  often  observed,  which  are  especially  noticeable  if  the  organism  is 
grown  in  mineral  media,  as  those  of  Conn  and  Naegeli.  The  organism 
does  not  liquefy  gelatine  and  grows  freely  on  all  the  usual  laboratory 
media  at  room  temperature,  the  dark  purplish  blue  or  in  some  cases 
brownish  coloration  of  the  medium  being  very  characteristic,  but 
this  freedom  of  growth  becomes  less  as  the  temperature  advances  to 
37°  C.,  and  the  cultures  themselves  die  at  40°.  ,The  .reaction  is 
invariably  alfolinp  althrmorh  the  medium  itself  may  have  been  in  the 
first  place  acid.  It  stains  well  with  all  the  ordinary  stains  but  does 
not  hold  the  Gram.  In  milk  the  bluish  tint  would  appear  to  be 
dependent  upon  certain  unknown  conditions,  and  in  the  sterile  milk 
used  for  the  laboratory  purposes  it  is  not  easy  to  obtain  it. 

Yellow_Milh — Bacillus  synxanthus  is  bpld  rp>apm|gffifo  for  curdling 
the  milk,  and  then  at  a  later  stage,  in  redissolving  the  curd,  produces 
a  yellow  pigment. 

In  addition  to  the  bacteria  of  fermentation  occasionally  present 
in  milk,  there  is  a  group  of  Various  Unclassified  Bacteria.  In 
milk  this  is  a  comparatively  small  group,  for  it  happens  that 
those  bacteria  in  milk  which  cannot  be  classified  as  fermentative  or 
pathogenic  are  few.  B.  coli  communis  occurs  here  as  elsewhere, 
and  might  be  grouped  with  the  gaseous  fermentative  organisms  on 
account  of  its  extraordinary  power  of  producing  gas  and  breaking  up 


202  BACTERIA  IN  MILK   AND  MILK  PRODUCTS 

the  medium  (whether  agar  or  cheese)  in  which  it  is  growing.  What 
its  exact  rdle  is  in  milk  it  would  be  difficult  to  say.  It  may  act,  as 
it  frequently  does  elsewhere,  by  association  in  various  fermentations. 
Some  authorities  hold  that  its  presence  in  excessive  numbers  may 
cause  epidemic  diarrhoea  in  infants  (Delepine). 

Several  years  ago  a  commission  was  appointed  by  the  British 
Medical  Journal  to  inquire  into  the  quality  of  the  milk  sold  in  some 
of  the  poorer  districts  of  London.  Every  sample  was  found  to 
contain  B.  coli,  and  it  was  declared  that  this  particular  microbe 
constituted  90  per  cent,  of  all  the  organisms  found  in  the  milk.* 
We  record  this  statement,  but  accept  it  with  some  reserve.  The 
diagnosis  of  B.  coli  eight  or  nine  years  ago  was  not  such  a  strict 
matter  as  to-day.  Still,  undoubtedly,  this  particular  organism  is  not 
uncommonly  found  in  milk,  and  its  source  is  uncleanly  dairying.  In 
the  same  investigation,  Proteus  vulgaris,  B.  fluorescens,  and  many 
liquefying  bacteria  were  frequently  found.  Their  presence  in  milk 
means  contamination  with  putrefying  matter,  surface  water,  or  a 
foul  atmosphere. 

nnd  their  way  into   milk  .in..  j 
" 


practice  of  adulterationimrTolil  bjT^pT^SH"  dirty  dairies  and  milk 
shops,  afford  ample  opportunity  for  aerial  pollution. 

Another  unclassified  group  occasionally  present  in  milk  is  repre- 
sented by  TTVl]]^,  particularly  Oidium  lactis,  the  mould  which  causes 
a  white  fur,  possessing  a  sour  odour.  It  is  allied  to  the  Mycoderma 
albicans  (0.  albicans),  which  also  occurs  in  milk,  and  causes  the 
whitish-grey  patches  on  the  mucous  membrane  of  the  mouths  of 
infants  (thrush).  These  and  many  more  are  occasionally  present  in 
milk. 

2.  THE  DISEASE-PKODUCING  POWER  OF  MILK 

The  general  use  of  milk  as  an  article  of  diet,  especially  by  the 
younger  and  least  resistant  portion  of  mankind,  very  much  increases 
the  importance  of  the  question  as  to  how  far  it  acts  as  a  vehicle  of 
disease.  Eecently,  considerable  attention  has  been  drawn  to  the 
matter,  though  it  is  now  a  number  of  years  since  milk  was  proved  to  be 
a  channel  for  the  conveyance  of  infectious  diseases.  During  the  last 
twenty  years,  particular  and  conclusive  evidence  has  been  deduced 
to  show  that  milch  cows  may  themselves  afford  some  measure  of 
infection.  The  extensive  work  on  tuberculosis  by  three  Royal  Com- 
missions has  done  much  to  obtain  new  light  on  the  conveyance  of 
that  disease  by  milk  and  meat.  The  enormous  strides  in  the  know- 
ledge of  the  bacteriology  of  diphtheria  and  other  germ  diseases  have 
also  placed  us  in  a  better  position  respecting  the  conveyance  of  such 
diseases  by  milk.  Generally  speaking,  for  reasons  already  given, 
*  Brit.  Med.  Jour.,  1895,  vol.  ii.,  p.  322. 


MILK-BORNE  TUBERCULOSIS  203 

milk  affords  an  ideal  medium  for  bacteria,  and  its  adaptability  there- 
fore for  conveying  pathogenic  organisms  is  undoubted.  We  shall 
speak  shortly  of  the  outstanding  facts  of  the  chief  diseases  carried  by 
milk. 

Milk-borne  Tuberculosis 

It  is  a  well-known  fact  that  tuberculosis  is  a  common  disease  of 
cattle.  Probably  not  less  than  20  to  30  per  cent,  of  milch  cows  in 
this  country  are  affected  with  it.  Therefore,  at  first  sight  it  might 
appear  that  the  consumption  of  milk  from  such  animals  would  lead 
to  considerable  spread  of  the  disease.  But  in  point  of  fact  there  are 
two  limiting  conditions.  The  first  has  relation  to  the  o^u^stioji.  of 
the  CQinmunicability  of  the  disease  from  the  cow  to  man.  The 
second  concerns  the  degree  of  disease  in  the  cow  which  can  affect  the 
milk.  It  is  necessary  that  both  points  should  be  discussed  some- 
whaTfully.  But  the  former  question  will  be  discussed  in  the  chapter 
dealing  with  Tuberculosis  (see  pp.  338-346).  The  latter  condition 
only  will  be  discussed  here.  It  has  reference  to  that  which  limits  the 
transmissibility  of  tuberculosis  from  the  cow  to  man  by  means  of 
milk  has  relation  to  the  well-established  fact  that  the  milk  of  tuber- 
culous cows  is  only  certainly  infective  when  the  tuberculous  disease 
affects  the  udder*  This  is  not  necessarily  a  condition  of  advanced 
tuberculosis.  The  udder  may  become  infected  at  a  comparatively  early 
stage.  The  presence  or  absence  of  tubercle  bacilli  in  the  milk  of  cows 

*  This  is  the  generally  accepted  view,  but  it  should  be  added  that  various 
workers  have  shown  that  cows  having  generalised  tuberculosis,  but  apparently  un- 
affected udders,  may  yield  tuberculous  milk.  Quite  recently  (1903)  Mohler,  as  the 
result  of  a  long  series  of  experiments,  arrives  at  the  following  important  con- 
clusions : — (1)  That  the  tubercle  bacillus  may  be  demonstrated  in  milk  from  tuber- 
culous cows  when  the  udders  show  no  perceptible  evidence  of  disease  either 
macroscopically  or  microscopically ;  (2)  that  the  bacillus  of  tuberculosis  may  be 
excreted  from  such  an  udder  in  sufficient  numbers  to  produce  infection  in  experi- 
mental animals,  both  by  ingestion  and  inoculation  ;  (3)  that  in  cows  suffering  from 
tuberculosis  the  udder  may,  therefore,  become  affected  at  any  moment ;  (4)  that  the 
presence  of  the  tubercle  bacillus  in  the  milk  of  tuberculous  cows  is  not  constant,  but 
varies  from  day  to  day  ;  (5)  that  cows  secreting  virulent  milk  may  be  affected  with 
tuberculosis  to  a  degree  that  can  be  detected  only  by  the  tuberculin  test;  (6)  that 
the  physical  examination  or  general  appearance  of  the  animal  cannot  foretell  the 
infectiveness  of  the  milk ;  (7)  that  the  milk  of  all  cows  which  have  reacted  to  the 
tuberculin  test  should  be  considered  as  suspicious,  and  should  be  subjected  to 
sterilisation  before  using ;  and  (8)  that  it  would  be  better  still  that  tuberculous  cows 
should  not  be  used  for  general  dairy  purposes.*  Experiments  by  Lydia  Rabino- 
witsch  have  given  somewhat  similar  results.  She  relies  on  tuberculin  as  a  test  of 
infectivity,  and  the  animal  experiment  as  proof  of  tuberculosis  in  milk.f  Ravenel 
also  maintains  that  cows  which  show  no  evidence  of  tuberculous  udders,  but  which 
react  to  tuberculin,  may  yield  tubercle  bacilli  in  their  milk.  J.  H.  Young  of  Aberdeen 
maintains,  on  the  other  hand,  that  cows  free  from  udder  disease  though  reacting  to 
tuberculin  yield  milk  free  from  tuberculosis.  £ 

*  Bureau  of  Animal  Industry,  Washington,  U.S.A.,  Bulletin  44,  1903,  p.  93. 
t  See  also  Zeitschrift  fur  Thiermedicin,  1904,  p.  202, 
+  Brit.  Med,  Jour,,  1903,  i.,  p.  816, 


204  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

with  udder  tuberculosis  is  greatly  dependent  on  the  extent  of  the  dis- 
ease in  the  udder.  But  to  make  the  milk  infective  the  udder  must  be 
affected,  and  milk  from  such  an  udder  possesses  a  considerable  degree 
of  virulence.  When  the  udder  is  thus  itself  the  seat  of  disease,  not 
only  the  derived  milk,  but  the  skimmed  milk,  butter-milk,  and  even 
butter,  may  all  contain  tuberculous  material.  Furthermore,  tuber- 
cular disease  of  the  udder  spreads  in  extent  and  degree  with  extreme 
rapidity.  From  these  facts  it  will  be  obvious  that  it  is  of  first-rate 
importance  to  be  able  to  diagnose  udder  disease.  This  is  not  always 
possible  in  the  early  stage.  The  signs  upon  which  most  reliance  may 
be  placed  are  the  enlargement  of  the  lymph-glands  lying  above  the 
posterior  region  of  the  udder ;  the  serous,  yellowish  milk  which  later 
on  discharges  small  coagula ;  the  partial  or  total  lack  of  milk  from 
one  quarter  of  the  udder  (following  upon  excessive  secretion) ;  the 
hard,  diffuse  nodular  swelling  and  induration  of  a  part  or  the  whole 
wall  of  the  udder ;  and  the  detection  in  the  milk  of  tubercle  bacilli. 
The  whole  organ  may  increase  in  weight  as  well  as  size,  and  on  post- 
mortem examination  show  an  increase  of  connective  tissue,  a  number 
of  large  nodules  of  tubercle,  and  a  scattering  of  small  granular  bodies, 
known  as  "miliary"  tubercles. 

The  udder  is  affected  in  about  2  per  cent,  of  the  cows  in  the 
milking  herds  in  this  country  (MacFadyean).*  In  London  about 
0*2  per  cent,  of  the  cows  are  so  affected,  as  judged  by  clinical 
observation.  It  will  be  remembered  that  in  the  country  generally 
between  20-30  per  cent,  of  the  cows  suffer  from  tuberculosis.  In 
London  15-20  per  cent,  are  tuberculous.  The  higher  standard  in 
London  is  due  to  better-class  animals  being  housed  in  London,  to 
more  thorough  inspection,  and  to  the  fact  that  there  is  no  inbreeding. 
But  let  us  take  the  generally-accepted  figure  of  2  per  cent,  of  the 
cows  in  the  United  Kingdom  as  having  tuberculous  udders,  and 
therefore  yielding,  in  greater  or  less  degree,  tuberculous  milk,  leaving 
altogether  out  of  account  cows  which  may  be  tuberculous  but  are 
not  affected  in  the  udder.  In  the  United  Kingdom  in  1901  there 
were  4,102,000  milch  cows.f  If  we  take  2  per  cent,  of  these  as  having 
tuberculous  udders,  it  gives  us  80,000.  The  average  annual  yield  of 
milk  per  cow  may  be  taken  as,  at  least",  400  gallons,!  which  means  that 
from  these  80,000  tuberculous  udders  32,000,000  gallons  of  milk  are 
obtained.  It  is  not  asserted  that  this  large  amount  of  milk  is  actually 
virulent  with  tuberculous  matter,  but  it  will  be  admitted  that  the 
entire  amount  of  it  is  open  to  grave  suspicion,  if  not  absolute  con- 
demnation. 

*  Trans,  of  Brit.  Congress  on  Tuberculosis,  1902,  vol.  i.,  p.  84. 
t  In  the  United  States  of  America  there  were  in  1901,  18,112,000  railch  cows  in 
actual  dairy  use. 

$  420  gallons  is  the  generally  accepted  figure. 


PLATE  18. 


Bacillus  tuberculosis.     Film  preparation  from  glycerine  glucose,  agar  culture.     Four  months  old — 
2  months  at  37°  C.,  and  2  months  at  20°  C.    Stained  with  carbol  fuchsin.     x  1000. 


?^«5fft 


Bacillus  tuberculosis,  in  Udder  of  Cow.    Stained  with  carbol  fuchsin  and  methylene  blue. 

x  1000. 


[7o  /ace  pafire  204. 


MILK-BORNE  TUBERCULOSIS 


205 


There  are  a  variety  of  conditions  in  addition  to  the  vera  causa,  the 
presence  of  the  bacillus  of  tubercle,  which  make  the  disease  common 
amongst  cattle.  Constitution,  temperament,  age,  work,  food,  in- 
breeding, and  prolonged  lactation,  are  the  individual  features  which 
act  as  predisposing  conditions ;  they  may  act  by  favouring  the  pro- 
pagation of  the  bacillus  or  by  weakening  the  resistance  of  the  tissues. 
To  this  category  must  further  be  added  conditions  of  environment. 
Rid  stabling,  dark,  ill-ventilated  stalls,  high  temperature,  prolonged 
and  close  contact  with  other  cows,  all  tend  in  the  same  direction. 

The  danger  from  drinking  raw  tuberculous  milk  only  exists  for 
persons  who  use  it  as  their  sole  or  principal  food,  that  is  to  say,  young 
children  and  certain  invalids.  With  adults  in  normal  health,  the 
danger  is  greatly  minimised,  as  the  healthy  digestive  tract  is  relatively 
insusceptible.  Moreover,  dairy  milk  is  almost  invariably  mixed  milk ; 
that  is  to  say,  that  if  there  is  a  tubercular  cow  in  a  herd  yielding  tubercle 
bacilli  in  her  milk,  the  addition  of  the  milk  of  the  rest  of  the  herd  so 
effectually  dilutes  the  whole  as  to  render  it  in  some  degree  innocuous. 

It  should  not  be  forgotten  that  milk  may  become  Jjubercular 
through  the  carelessness  or  dirty  habits  of  the  milker.  Such  a 
common  practice  as  moistening  the  hands  with  saliva  previously  to 
milking  may,  in  cases  of  tubercular  milkers,  effectually  contaminate 
the  inilk.  Again,  it  may  become  polluted  by  dried  tubercular  matter 
getting  into  it  from  dust  or  infected  dried  excreta.  Such  convey- 
ances must  be  of  rare  occurrence,  yet  their  possibility  should  not  be 
forgotten. 

The  Tubercle  Bacillus  in  Market  Milk. — Investigations  have  been 
made  in  many  cities  as  to  the  actual  occurrence  of  the  tubercle 

TABLE  showing  the  Total  Number  of  Milks  Examined  Bacteriological  li/  for 

Tubercle  Bacilli  from  August  1896  to  31^  December  1903. 

(Liverpool.) 


Total 

Town  Samples. 

Country  Samples. 

number 

of  Samples 

taken. 

Number 
taken. 

Tubercular. 

Percentage 
Tubercular. 

Number 
taken. 

Tubercular. 

Percentage 
Tubercular. 

1896 

119 

83 

4 

4-8 

36 

5 

14'0 

1897 

150 

63 

4 

6-3 

87 

5 

5*7 

1898 

112 

84 

7 

8-3 

28 

5 

17-9 

1899 

352 

167 

1 

0-6 

185 

15 

8-1 

1900 

560 

255 

4 

1-5 

305 

5 

1-6 

1901 

566 

254 

2 

0-7 

312 

20 

6'4 

1902 

595 

213 

1 

0'4 

382 

32 

8-3 

1903 

582 

231 

2 

0-8 

351 

19 

5'6 

bacillus  in  milk  as  placed  on  the  market.     It  has  been  found  in 

r: 

Or  rut    ' 


206  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

varying  percentage,*  but,  as  a  rule,  the  milk  coming  in  to  the 
cities  from  the  country  has  contained  more  tubercle  bacilli  than 
the  milk  obtained  from  the  town  cows.  This  characteristic  has 
been  found  to  occur  in  London,  Manchester,  Liverpool,  and  other 
cities. 

It  should  also  be  added  that  there  are  a  number  of  cases  on  record 
where  the  tubercle  bacillus  has  been  found  in  butter. 

The  Virulence  of  the  Tubercle  Bacillus  in  Milk. — Martin  and 
Woodhead  concluded  as  the  result  of  their  investigations  for  the 
Koyal  Commission  on  Tuberculosis  that  tuberculous  milk  possessed 
a  high  degree  of  virulence  for  man.  Sir  Eichard  Thome  held  that 
tabes  mesenterica  (alimentary  tuberculosis)  of  children  had  not 
declined  as  phthisis  (pulmonary  tuberculosis)  had  done  in  recent 
years  on  account  of  the  conveyance  of  the  virus  of  tubercle  in  milk.f 
If  the  occurrence  of  primary  lesion  in  the  intestine  is  indication  of 
infection  through  the  alimentary  tract,  then  it  is  instructive  to  notice 
that  of  all  the  tuberculosis  in  children  in  this  country  about  25  per 
cent,  is  alimentary  in  origin,  and  in  60  to  70  per  cent,  of  the  cases 
the  mesenteric  glands  are  affected.  Both  of  these  figures  deal  with 
deaths  only,  but  as  Kaw  has  pointed  out,  no  doubt  a  number  of  cases 
of  alimentary  tuberculosis  recover,  the  infection  having  been  mild. 
Amongst  269  tuberculous  children  under  twelve  years  of  age  whom 
Dr  Still  examined  post  mortem,  he  found  it  possible  to  determine 
the  channel  of  infection  with  some  degree  of  certainty  in  216 
cases.  In  138  (63*8  per  cent.)  infection  entered  through  the  lung ; 
in  63  (29'1  per  cent.)  primary  infection  occurred,  in  all  prob- 
ability through  the  intestine.  Of  children  up  to  two  years  of  ago 
he  found  65  per  cent,  contracted  infection  through  the  lung,  and 
22  per  cent,  through  the  intestine.  In  infants  under  one  year  of 
age  apparently  only  13  per  cent,  contracted  tuberculosis  through  the 
intestine.  { 

It  is  recognised  that,  owing  to  the  great  tendency  to  generalisa- 
tion of  tuberculosis  in  children,  it  is  a  matter  of  extreme  difficulty 
to  determine  which  was,  in  fact,  the  primary  channel  of  infection, 
and  this  must  be  taken  into  consideration  in  estimating  the 
significance  of  the  frequency  in  the  above  figures.  It  should  also 
be  remembered  that  the  tubercle  bacillus  may,  and  probably 

*  In  1898,  14  per  cent,  of  the  milks  examined  in  Berlin  by  Petri  contained  the 
tubercle  bacillus.  In  1899,  in  Islington,  the  percentage  was  14 '4;  in  1893,  St 
Petersburg,  5  percent. ;  in  1901,  in  London,  7  per  cent.  (Klein) ;  in  1901,  at  Croydon, 
67  per  cent.,  and  Manchester,  9'5  per  cent. ;  in  1902,  at  Woolwich,  10  per  cent.  ; 
in  Camberwell,  11  per  cent.  ;  in  the  City  of  London  and  in  Finsbury,  nil.  These 
percentages  must  not  be  accepted  as  anything  but  passing  figures  and  illustrations 
of  what  various  investigators  have  found  under  varying  conditions. 

f  The  Administrative  Control  of  Tuberculosis  (Harben  Lectures),  1899,  pp.  5-7 
and  28-32. 

$  Practitioner,  1901  (July),  p.  94. 


MILK-BORNE  TYPHOID  207 

does,  pass  through  the  intestinal  wall  into  the  nearest  lymphatic 
glands,  leaving  no  visible  trace  on  the  intestine.  Further,  owing 
to  the  fact  that  children  swallow  their  pulmonary  expectoration, 
secondary  infection  of  the  intestine  may  rapidly  follow  primary 
infection  of  the  lungs.  Hence  it  comes  about  that,  in  many  cases, 
the  intestine  and  mesenteric  glands  are  affected,  and  yet  such  a 
condition  cannot  be  taken  as  evidence  of  the  infection  by  food.  Dr 
Still  concludes  that  (a)  the  commonest  channel  of  infection  with 
tuberculosis  in  childhood  is  through  the  lung ;  (b)  infection  through 
the  intestine  is  less  common  in  infancy  than  in  later  childhood; 
(c)  milk,  therefore,  is  not  the  usual  source  of  tuberculosis  in  infancy ; 
and  (d)  inhalation  is  much  the  commonest  mode  of  infection  in  the 
tuberculosis  of  childhood,  and  especially  in  infancy.  Dr  Still  has 
placed  on  record  5  cases  of  tuberculous  ulcer  of  the  stomach  in 
children. 

Taking  a  broad  view  of  the  facts,  it  would  appear  that  whilst 
tuberculosis  is  not  chiefly  spread  by  means  of  milk,  there  is 
unmistakable  evidence,  derived  from  pathological  and  clinical 
experience,  proving  that  tuberculous  milk  can,  and  does  on  occasion, 
set  up  some  form  of  tuberculosis  (bovine  or  human)  in  the  bodies 
of  man  and  other  animals  consuming  the  milk. 


Milk-borne  Typhoid  Fever 

Dr  Michael  Taylor  of  Penrith  was  the  first  to  establish  the  now 
well-known  fact  that  milk  may  act  as  a  vehicle  of  the  virus  of 
enteric  fever.  That  was  in  1857.*  Since  that  date  more  than  160 
epidemics  of  this  disease  have  been  traced  to  a  polluted  milk  supply. 
Schuder  states  that  17  per  cent,  of  all  typhoid  epidemics  are  due  to 
the  consumption  of  infected  milk. 

The  steps  in  the  process  of  infection  are  briefly  as  follow.  Enteric 
fever  affects  the  intestine,  and  hence  the  excreta,  especially  in  the 
early  stages  of  the  disease,  are  charged  with  large  numbers  of  the 
causal  bacilli.  It  is  now  known  that  the  sweat,  expectoration  from 
the  lungs,  and  the  urine  of  a  typhoid  patient  may  also  contain  the 
typhoid  bacillus.  Indeed,  the  urine  in  25  per  cent,  of  the  cases 
generally  contains  large  numbers  of  the  bacillus  (Horton  Smith).-]* 
There  is  also  evidence  to  show  that  the  bacilli  may  remain  in  the 
urine  for  long  periods  after  convalescence,  even  for  months  and 
possibly  for  years.  The  bowel  discharges  and  the  urine  are,  therefore, 
the  two  chief  channels  by  which  the  typhoid  bacillus  is  excreted. 
It,  therefore,  readily  gains  access  to  the  soil,  to  drains,  and  eventually 

*  Edin.  Med.  Jour.,  1858,  pp.  993-1004. 
t  Lectures  on  Typhoid  Fever,  1900. 


208  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

on  occasion  to  the  water  supply,  and  thus  into  milk  and  back  again 
to  man.  The  virus  does  not  always  pass  in  the  discharges  to  water 
and  milk,  but  may  reach  them  by  becoming  dried  dust.  A  small 
pollution  may  in  this  way  set  up  widespread  disease.  (For  the 
behaviour  of  the  typhoid  bacillus  in  soil,  see  pp.  145-150.) 

The  most  common  way  for  milk  to  become  infected  by  the 
typhoid  bacillus  is  through  infected  water.  Such  water  may  be 
added  to  the  milk  by  way  of  adulteration  or  by  accident;  or  the 
milk  vessels  may  have  been  "  cleansed "  with  polluted  water  (in  29 
per  cent,  of  milk-borne  outbreaks  according  to  Schuder).  Another 
source  of  infection  of  the  milk  is  when  persons  suffering  from  a 
mild  attack  of  typhoid  fever  continue  to  work  a  dairy  or  otherwise 
deal  with  milk,  and  this  has  proved  a  frequent  means  of  infection. 
Flies  doubtless  convey  the  germ  of  the  disease  not  infrequently,  as 
was  shown  in  the  Spanish- American  War  of  1898  *  and  the  Boer 
War  of  1900-1901.1 

Though  the  typhoid  bacillus  appears  not  to  have  the  power  of 
rapid  multiplication  in  milk,  it  has  the  faculty  of  existing  in  milk 
for  a  considerable  time  (twenty  days  or  longer)  even  when  milk  has 
curdled  or  soured,  and  may  thus  infect  milk  products,  such  as  butter 
and  cheese.  But  infection  by  milk  products  may  be  eliminated  as 
of  too  rare  occurrence  to  deserve  attention.  The  bacillus  does  not 
coagulate  milk  like  its  ally  the  B.  coli  communis,  which  is  a  much 
more  frequent  inhabitant  of  milk.  It  flourishes  in  milk  at  room 
temperature  and  blood-heat,  and  does  not  produce  acid  or  alter  the 
appearance  of  the  milk. 

Several  typical  milk-borne  outbreaks  of  typhoid  fever  may  be 
cited : — 

1.  Infection  from  Personal  Contact  with  Typhoid  Patients. — At 
Penrith,  it  appears  that  about  the  beginning  of  September,  1857,  a 
young  servant  girl,  E.  0.,  returned  home  to  Penrith  from  Liverpool 
suffering  fom  typhoid  fever.  The  family  of  which  she  was  a 
member  consisted  of  father,  mother,  and  five  children,  of  whom  she 
was  the  eldest.  The  cottage  in  which  they  lived  consisted  of  two 
ill-ventilated  and  ill-lighted  rooms,  a  kitchen  or  sitting-room,  and  a 
bedroom  opening  out  of  it.  The  father  possessed  three  cows,  and 
carried  on  a  small  milk  business  dealing  with  some  fourteen 
families.  The  mother  'milked  the  cows,  and  the  milk  was  brought 
into  the  kitchen,  direct  from  the  byre,  and  in  due  course  dis- 
tributed in  tin  measures  amongst  the  customers.  After  her  return 
home  the  girl  continued  ill  for  about  a  fortnight,  during  which 
period  she  was  nursed  by  her  mother  in  the  kitchen  or  common 

*  American  War  Department,  Official  Report,  1900. 

t  Brit.  Med.  Jour.,  1901,  i.,  p.  642  et  seq.  ;  ibid.,  1902,  ii.,  pp.  936-941  (Firth 
and  Horrocks). 


MILK-BORNE  TYPHOID  209 

sitting-room.  At  the  end  of  the  fourth  week  in  September  she  was 
convalescent,  and  began  to  help  at  once  in  the  distribution  of  the 
milk.  Two  other  children  of  the  family  sickened  and  passed  through 
the  fever.  The  mother  nursed  all  three  patients,  and  continued  to 
milk  the  cows  and  attend  to  the  distribution  of  the  milk.  In 
October  and  November  some  13  cases  of  typhoid  fever  occurred 
in  seven  families  dealing  with  the  infected  cottage,  and  from  these 
primary  cases  a  number  of  persons,  over  a  somewhat  wide  area,  were 
infected  by  contact.  By  most  careful  observation  and  reasoning,  Dr 
Taylor  arrived  at  the  conclusion  that  the  milk  became  contaminated 
in  the  kitchen  of  this  cottage,  from  the  typhoid  patients  there 
being  nursed.* 

2.  Infection  from  Washing  Milk  Vessels  with  Polluted  Water. — At 
Clifton,  Bristol,  in  October  1897,  an  outbreak  affected  244  persons, 
31  of  whom  died.    Ninety-six  per  cent,  of  the  patients  consumed  sus- 
pected milk.    It  happened  that  a  brook  received  the  sewage  of  thirty- 
seven  houses,  the  overflow  of  a  cesspool  serving  twenty-two  more,  the 
washings  from  fields  over  which  the  drainage  of  several  others  was 
distributed,  and  the  direct  sewage  from  at  least  one  other,  and  then 
flowed  directly  through  a  certain  farm.     In  September  it  seems  that 
some  excreta  from  a  man  suffering  from  typhoid  fever  gained  access 
to  the  brook.     The  water  of  this  stream  supplied  the  farm  pump,  and 
the  water  itself,  it  is  scarcely  necessary  to  add,  was  highly  charged 
with   putrescent  organic  matter  and  micro-organisms.     This  water 
was   used   for  washing   the  milk-cans   from   this   particular  farm, 
otherwise  the  dairy  arrangements  were  efficient.     Part  of  the  milk 
was  distributed  to  fifty-seven  houses   in   Clifton ;    in  forty-one   of 
them   cases   of  typhoid  occurred.     Another  part  of  the  milk  was 
sold   over   the  counter;    twenty  households   so  obtaining  it  were 
attacked  with  typhoid  fever,  and  a  number  of  further  infections 
arose  in  the  course  of  a  third  delivery.-)- 

3.  Infection  from   Water  added  to  Milk. — At  Moseley,  in  1873, 
96   persons  in  fifty  families  contracted  typhoid  fever  from  milk. 
Boy  at  milkman's  house  fell  ill  of  typhoid  fever,  suffered  there  for  a 
fortnight  and  died.    Two  wells  were  polluted  from  a  privy  into  which 
typhoid  excreta  had  been  thrown.     The  water  of  the  well  was  added 
accidentally  or  intentionally  to  the  milk.     Dr  Ballard  summed  up 
his  view  of  the  causation  in  this  outbreak  as  follows : — (1)  Two 
wells   upon  adjoining  premises   occupied   by   milk   sellers   became 
infected  early  in  November  with  the  infectious  matter  or  virus  of 
enteric    fever,   through   the   soakage   from   a  privy  into   them   of 
excremental    matters    containing    that   matter    of   infection.       (2) 
Through  the  medium  of  water  drawn  from  these  wells  the  milk 

*  Edin.  Med.  Jour.,  1858,  pp.  993-1004. 

t  Trans.  Epidem.  Soc.  of  London,  vol.  xvil,  pp.  78-103  (Dr  D.  S.  Davies). 

O 


210  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

supplied  by  these  milk  sellers  became  infected,  and  many  of  their 
regular  customers  who  drank  the  milk  suffered  from  the  disease. 
(3)  The  same  infected  milk  having  been  sold  to  two  other  milk 
purveyors,  some  of  the  persons  using  the  milk  supplied  by  these 
milkmen  also  suffered  in  a  similar  manner.  (4)  There  is  no 
evidence  that  the  disease  spread  in  these  districts  in  any  other 
way  than  through  this  milk.* 

4.  Infection  from   the    Air    ly    Dried    Typhoid   Excreta.  —  At 
Millbrook,  in  Cornwall,  in   1880  (July — September),  an  outbreak 
occurred  having  a  total  number  of  cases  of  91.     In  this  instance  part 
of  a  slaughter-house  not  used  as  such  but  as  a  wash-house  was 
boarded  off  to  constitute  a  dairy.     On  a  shelf  of  this  dairy  the  milk 
was  habitually  set  in  pans,  exposed  to  the  air.     In  one  corner  of  the 
slaughter-house,  nearest  the  dairy,  was  a  badly  trapped  and  very 
offensive  drain  inlet.     Close  to  this  inlet  ran  the  wooden  partition 
between  the  slaughter-house  and  the  dairy,  which  near  the  inlet  had 
been  long  broken  away.     The  drain  was  in  communication  with  an 
old  square  drain  which  had  received  typhoid  excreta,  so  that  the 
infected  sewer  air  from  the  inlet  had  free  access  to  the  dairy  and  the 
exposed  milk  which  stood  in  the  dairy.     There  was  evidence  to  show 
that  the  drain  was  in  a  dry  and  "  waterless  "  condition.     Six  cases  of 
typhoid   occurred  in  the  butcher's   family.f      A  similar   outbreak 
occurred  in  county  Durham  in  1893. 

5.  Infection  from  Contaminated  Cloths  and  Clothes. — At  Barrow- 
ford  in  Lancashire  there  occurred  a  typhoid  epidemic  in  1876.     The 
total  number  of  cases  was  57,  all  of  whom  drank  the  suspected  milk. 
The  farmer  had  had  typhoid  fever  in  his  house  for  two  or  three 
weeks  before  the  outbreak,  and  no  precautions  had  been  taken  to 
prevent  the  spread  of  the  disease.     The  milk  was  left  for  some  time 
in  the  farm-house  before  being   sold.     The   milk-tins  were  wiped 
with  the  same  "  dish  cloth  "  as  that  used  among  the  fever  patients. 
The  farmer  himself  nursed  his  children,  and  then  went  immediately 
without  disinfection  amongst  his  cattle  and  milked  them  in  the  same 
clothes  he  had  worn  whilst  nursing  his  children.     The  cases  occurred 
within  a  very  short  space  of  time,  and  every  one  of  them  without 
exception   drank   the  milk   from   this   farm.      Twenty-five   of   the 
patients  were  under  ten  years  of  age.     There  was  no  other  typhoid 
in  the  district. 

6.  Infection   owing  to   Cooling  Milk  in   Water. — At  Springfield, 
Mass.,  U.S.A.,  in  1892,  an  outbreak  affecting  150  persons  (25  of 
whom  died).     Upon  the  farm  supplying  the  implicated  milk  there 
was  one,  and  probably  more  than  one,  case  of  typhoid  fever.     The 
farmer  submerged  his  sealed  milk-cans  when  full  of  milk,  in  the  well 

*  Report  of  Local  Government  Board,  1874,  p.  92. 
t  Brit.  Med.  Jour.,  1881,  i.,  p.  20. 


MILK-BORNE  DIPHTHERIA  211 

adjoining  the  cow  yards,  with  the  object  of  keeping  the  milk  cool. 
The  water  in  this  was  polluted,  and  it  was  found  that  four  of  nine 
milk-cans  leaked  when  inverted.  Hence  it  became  evident  that 
water  could  gain  access  if  the  cans  were  submerged  as  they  had 
been.  The  investigators  suggest  that  as  the  typhoid  excreta  of  the 
patient  were  placed,  undisinfected,  in  the  privy,  and  the  contents  of 
the  latter  spread  over  the  tobacco  field,  the  germs  of  typhoid  may 
have  gained  access  to  the  well  by  dirt  from  the  labourers'  boots,  who 
both  worked  in  the  field  and  at  the  milk.  Coliform  organisms  were 
found  in  the  well  water.* 


Milk-borne  Diphtheria 

Milk  is  a  favourable  medium  for  the  B.  diphtherice.  The  organism 
both  lives  and  multiplies  in  ordinary  sterilised  milk,  but  it  thrives 
better  in  milk  at  comparatively  low  temperatures  than  at  37°  C.  In 
ordinary  milk,  unsterilised  and  unprepared,  the  commoner  organisms 
multiply  much  more  rapidly,  and  so  the  diphtheria  bacillus  is  in 
all  probability  soon  crowded  out. 

The  cases,  however,  in  which  the  B.  diphtherice  has  been  actually 
isolated  from  market  milk  are  extremely  few.  In  the  outbreak  of 
diphtheria  at  Senghenydd  in  South  Wales,  in  1899,  Bowhillf 
isolated  a  diphtheria  bacillus  from  the  suspected  milk.  The  culture 
of  the  bacillus  in  broth  proved  fatal  to  a  guinea-pig  in  two  days. 
In  the  same  year,  EyreJ  isolated  a  virulent  diphtheria  bacillus 
from  some  milk  implicated  in  an  outbreak  of  diphtheria  in  a 
school.  In  1900,  Klein  §  also  reported  the  isolation  of  a  genuine 
diphtheria  bacillus  in  an  examination  of  100  samples  of  milk  in 
London.  Lastly,  Dean  and  Todd,  isolated  the  B.  diphtherice  from  cow's 
milk  in  1901.11  These  are  the  only  four  authentic  cases  of  actual 
detection  of  the  B.  diphtherice  in  ordinary  milk  with  which  we  have 
met. 

There  is  a  question  which  must  now  be  considered,  viz. :  the 
relationship  existing  between  diphtheria  in  man  and  animals  and  the 
milk  supply.  How  does  the  milk  become  infected  ? 

(1)  In  the  first  place,  it  is  now  generally  held  that  the  B. 
diphtherice  has  a  comparatively  wide  distribution  in  nature ;  whilst 
it  appears  not  to  be  conveyed  by  water,  it  is  believed  that  certain 
conditions  of  soil  favour  its  growth  as  a  saprophyte.  But  this  is 
not  proved.  (2)  In  the  second  place,  it  has  been  proved  that  persons 

*  Boston  Med.  and  Surg.  Jour.,  1893,  ii.,  p.  485  (Sedgwick  and  Chapin). 
t  Veterinary  Record,  8th  April  1899,  No.  561.     Jour,  of  Stale  Medicine,  1899, 
pp.  705-710. 

J  Brit.  Med.  Jour.,  1899,  vol.  ii.,  p.  586. 
§  Jour,  of  Hygiene,  1901,  vol.  i.,  p.  85. 
||  Ibid.,  1902,  vol.  ii.,  pp.  194-205. 


212  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

suffering  from  diphtheria  are  foci  of  infection.  The  exact  channels 
of  infection  differ  under  varying  circumstances ;  but,  in  general,  the 
source  of  infection  is  the  throat  and  mouth  of  the  patient.  Anything 
which  comes  into  contact  with  the  mucous  membrane  becomes 
infected.  Thus  handkerchiefs,  sweets,  children's  toys,  etc.,  may  act 
as  the  vehicles  of  contagion.  The  mucus  and  saliva  may  also  be 
infective,  and  in  speaking,  kissing,  coughing,  or  expectorating  such 
mucus  (probably  rich  in  bacilli)  may  be  disseminated  in  very  fine 
particles,  and  so  carry  the  disease.  It  is  by  such  means  that  the 
disease  is  spread.  Moreover,  there  is  the  fact  of  the  long  period 
during  which  the  human  throat  may  remain  infective.  Professor 
Sims  Woodhead  has  recently  stated  that  the  persistence  of  the 
diphtheria  bacillus  for  periods  up  to  eight  weeks  is  of  very  common 
occurrence.  (3)  Kichardiere  and  Tollemer  *  and  others  have  proved 
that  the  dust  floating  in  the  air  of  a  diphtheria  ward  may  contain 
large  numbers  of  diphtheria  bacilli,  and  in  this  way  milk  and  other 
foods  may  become  contaminated. 

Between  1878  and  1883,  certain  outbreaks  of  diphtheria  due  to 
milk  appeared  to  suggest  that  the  cow  itself  might  suffer  from 
diphtheria.  The  discovery  of  the  Klebs-Loffler  bacillus  in  1883 
furnished  the  basis  for  experimental  work,  and  in  1886  Dr  Klein 
undertook  some  experiments  to  ascertain  whether  or  not  diphtheria 
was  inoculable  into  cows.  He  took  for  the  experiment  two  healthy 
milch  cows  which  had  calved  some  three  or  four  weeks  previously. 
One  c.c.  of  broth  culture  of  B.  diphtheria  (derived  from  human 
diphtheritic  membrane)  was  injected  under  the  skin  into  the  sub- 
cutaneous tissue  of  the  left  shoulder  in  each  of  the  two  cows.  Two 
or  three  days  after  the  inoculation  (a)  the  temperature  rose  (to 
40'6°),  and  the  animals  suffered  from  slight  general  malaise.  On 
the  third  day  (b)  a  tumour  appeared  at  the  site  of  inoculation,  which 
steadily  increased  in  size  to  the  seventh  day.  On  the  fifth  day  (c) 
a  papular  eruption  appeared  on  the  udder  and  hind  teat.  In  addition 
to  the  papules  there  were  half  a  dozen  vesicles,  and  some  round 
patches  covered  with  brown  crusts.  The  process  of  eruption  was 
mature  by  the  eighth  day.  In  the  lymph  of  the  vesicles  and  pustules 
the  B.  diphtherias  could  be  demonstrated,  according  to  Klein,  both 
microscopically  and  by  culture.  He  therefore  concluded  that  the 
B.  diphtherice,  as  such,  inoculated  into  the  shoulder  of  the  cow,  was 
received  into  the  general  system  of  the  cow,  and  produced  its  effects 
not  in  the  viscera,  but  on  the  udder  as  a  specific  eruption,  and  that 
before  the  end  of  five  days  after  inoculation,  was  finally  excreted  in 
the  cow's  milk.  "  The  presence  of  the  diphtheria  bacillus,"  he  wrote, 
"  could  with  certainty,  by  microscopic  and  culture  observations,  be 
demonstrated  in  the  rnilk  of  the  cow  collected  under  all  precautions ; 
*  Gazette  des  Maladies  Infantile*,  1899,  No.  10. 


MILK-BORNE  DIPHTHERIA  213 

the  number  of  bacilli  present  on  that  day  in  the  milk  amounted  to 
32  per  c.c.  Scrapings  from  vesicles  on  the  sixth  day  were  inoculated 
into  two  calves,  which  then  suffered  from  a  like  disease."  * 

During  1890  and  1891,  Dr  Klein  repeated  these  experiments 
on  milch  cows,  and  in  two  further  instances,  out  of  six  cows,  an 
eruption  was  produced  on  the  udder  and  teats,  and  in  one  of  these 
positive  cases  the  B.  diphtheria  was  found  in  the  milk  about  a  week 
after  inoculation. 

It  must  be  admitted  that  positive  results  did  not  always  follow 
these  experimental  researches.  Loffler,  Abbott,  Bitter,  and  others, 
including  many  veterinarians,  criticised  the  experiments,  and  held 
that  there  was  no  conclusive  evidence  that  diphtheria  was  a  bovine 
disease.  Since  that  time  some  twenty  milk-diphtheria  outbreaks 
have  been  investigated,  with  the  result  that,  with  one  or  two 
exceptions,  the  infectivity  of  the  milk  was  certainly  derived  from 
human  sources  and  not  from  bovine.  In  the  Croydon  outbreak  in 
1890,  at  Worcester  in  1891,  and  at  Glasgow  in  1892,  evidence  was 
obtained  which  appeared  to  support  Klein's  views. 

Up  to  the  present  it  may,  however,  be  said  that  the  evidence 
forthcoming  points  in  the  direction  of  human  rather  than  bovine 
infection  as  the  origin  of  the  diphtheria  bacillus  in  milk. 

An  interesting  investigation  has  recently  been  made  by  Dean  and 
Todd,  respecting  a  small  outbreak  of  diphtheria  occurring  in  1901/f* 
In  this  outbreak  several  individuals  suffered  from  diphtheria,  and 
several  others  in  the  same  households  suffered  from  sore  throat, 
probably  diphtheritic.  These  individuals  obtained  their  milk  from 
two  cows  suffering  from  a  contagious  eruptive  disease  of  the  udder, 
from  which  Dean  and  Todd  isolated  a  bacillus  indistinguishable  from 
Klebs  Loffler  bacillus  of  diphtheria.  The  case  was  a  very  interesting 
one.  But  the  whole  matter  of  bovine  diphtheria  is  sub  judice. 

It  was  then  in  1878,  that  evidence  was  forthcoming  in  support 
of  the  view  that  diphtheria,  like  typhoid  fever,  might  on  occasion  be 
spread  by  means  of  milk.  In  that  year,  Mr  W.  H.  Power  made  an 
inquiry  into  an  outbreak  of  diphtheria  in  North  London,  chiefly  in 
Kilburn  and  St  John's  Wood.  There  were  as  many  as  264  persons 
attacked,  and  38  died.  The  infection  invaded  some  118  different 
households.  The  epidemic  was  most  severe  in  May  (first  four  weeks), 
when  about  190  cases  occurred.  The  outbreak  terminated  abruptly. 
The  area  infected,  and  time  of  infection,  clearly  showed  that  there 
was  some  factor  at  work  over  a  circumscribed  area,  and  operative 

*  See  A  Treatise  on  Hygiene  and  Public  Health  (Stevenson  and  Murphy),  vol.  ii., 
pp.  161-164  (Klein).  Also  Local  Government  Board  Report,  1889,  p.  167  et  seq. 

f  Jour,  of  Hygiene,  April  1902  (vol.  ii.,  No.  2,  p.  194).  Experiments  on  the 
relation  of  the  Cow  to  Milk  Diphtheria,  by  George  Dean,  M.B.,  and  Charles  Todd, 
M.D. 


214  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

during  a  limited  time.  There  was  no  antecedent  throat  illness,  and 
no  school  infection  or  contact  contagion  traceable.  The  houses  were 
sanitarily  good,  and  had  a  good  water  supply.  There  was  but  one 
thing  common  to  most  of  the  cases,  and  this  was  the  milk  supply. 
It  was  found  that  within  the  area  of  the  greatest  prevalence  of 
throat  illness,  about  one-fifth  of  the  households  were  supplied  by  a 
common  milk  supply.  The  incidence  of  the  disease  fell,  actually  and 
relatively,  upon  consumers  of  the  suspected  milk. 

Inquiry  into  the  milk  supply  elicited  no  evidence  of  human 
disease  pollution,  nor  contamination  by  water  or  air.  Nor  was  there 
any  definite  disease  of  cows  present  at  the  time  as  far  as  could  be 
judged.  But  by  a  process  of  exclusion,  Mr  Power  suggested  that 
"  there  may  have  been  risk  of  specific  fouling  of  milk  by  particular 
cows  suffering,  whether  recognised  or  not,  from  specific  disease." 

Since  that  time  there  have  been  some  30  outbreaks  of  milk-borne 
diphtheria.  In  most  cases  the  milk  appears  to  have  been  infected 
directly  by  persons  suffering  from  the  disease,  recognised  or  un- 
recognised. 

Milk-borne  Scarlet  Fever 

There  are  some  seventy  milk-borne  epidemics  of  scarlet  fever  on 
record,  and  yet  comparatively  little  is  known  as  to  the  bacteriology 
of  the  disease  (see  p.  296).  In  almost  all  the  outbreaks  which  have 
occurred  there  is  evidence,  more  or  less  conclusive,  that  persons 
suffering  from  scarlet  fever  have  been  concerned  in  milking  or  in  the 
distribution  of  milk.  But  in  1882  Mr  W.  H.  Power,  in  investigating 
an  outbreak  of  milk-borne  scarlet  fever  in  North  London,  came  to  the 
conclusion  that  the  cow  had  been  the  exciting  cause  of  the  epidemic, 
and  was  suffering  from  some  diseased  condition  which  could  convey 
to  the  milk  the  virus  of  scarlet  fever.*  In  1885  occurred  the 
"  Hendon  outbreak  "  of  scarlet  fever,  which  affected  North  London 
districts  and  Hendon.  After  inquiry,  it  was  believed  that  the  scarlet 
fever  in  these  districts  followed  the  consumption  of  milk  from  a 
particular  farm  at  Hendon.  Further,  in  these  four  districts  wherein 
scarlatina  had  shown  an  extravagant  incidence  upon  the  milkman's 
customers,  the  disease  had  begun  its  peculiar  incidence  about  the  end 
of  November  or  beginning  of  December.  In  one  of  those  districts 
(South  Marylebone),  scarlatina  continued  day  by  day,  and  with 
increasing  force  up  to  the  date  of  the  inquiry,  to  attack  the  customers 
of  the  retail  business.  In  two  other  districts  (Hampstead  and  St 
Pancras),  after  attacking  in  some  numbers,  for  a  few  days  at  the  end 
of  November  and  beginning  of  December,  the  customers  of  the  busi- 
ness, the  disease  showed  no  fresh  attacks  for  about  ten  days  (a  short 
but  clearly  defined  intermission),  and  then  about  the  middle  of 

*  Supplement  to  the  Report  of  Local  Government  Board,  1882,  p.  65. 


MILK-BORNE  SCARLET  FEVER  215 

December  attacked  them  again  in  larger  numbers,  and  continued  to 
do  so  up  to  date  of  inquiry. 

The  chief  facts  concerning  the  distribution  of  the  milk  may  be 
set  out  as  follows :  (a)  The  Marylebone  customers  suffered  at  the  end 
of  November  and  up  till  the  end  of  the  third  week  in  December.  (5) 
The  Hampstead  cases  occurred  in  two  groups,  one  small  group  at  the 
end  of  November  and  a  larger  group  in  the  latter  part  of  December. 
(c)  The  St  Pancras  customers  suffered  like  the  Hampstead  ones,  but 
in  a  less  degree.  They  obtained  milk  from  the  same  vendors,  (d) 
The  St  John's  "Wood  customers  did  not  suffer  until  after  the  end  of 
the  year,  (e)  The  few  persons  affected  at  Hendon  suffered  early  in 
December,  having  consumed  milk  which  had  been  returned  from 
Marylebone,  and  at  the  same  time  new  cases  of  scarlet  fever  ceased 
to  occur  in  Marylebone.  Examination  was  then  made  to  ascertain 
if  there  had  been  any  possible  infection  of  the  milk  to  explain  this 
incidence  and  intermission. 

When  Mr  Power  came  to  inquire  as  to  the  movements  of  the 
cows,  he  learned  that  on  15th  November  three  newly-calved  cows 
arrived  at  the  Hendon  farm  from  Derbyshire,  this  event  shortly  pre- 
ceding the  first  attack  of  scarlatina.  It  happened  that  these  three 
animals  were  placed  in  a  shed  by  themselves,  and  their  milk  was  dis- 
tributed in  part  to  South  Marylebone,  Hampstead,  and  St  Pancras, 
immediately  preceding  the  outbreak  of  scarlatina  in  those  districts. 
On  examination  it  was  found  that  the  implicated  cows  were  suffering 
from  some  kind  of  disease  of  the  udders,  which  had  spread  to  other 
cows  in  the  herd.  It  would  appear  that  the  diseased  condition,  what- 
ever it  was,  had  been  introduced  by  one  of  the  Derbyshire  cows,  and 
had  then  spread  through  various  sheds  at  the  Hendon  farm.  Mr  Power 
was  able  by  the  most  minute  inquiry  to  trace  the  movements  of  those 
cows  and  the  various  sheds  in  which  they  were  placed  from  time  to 
time,  and  he  held  that  the  various  recrudescences  of  the  outbreak  in 
North  London  corresponded  with  the  movements  of  the  affected  cows. 

The  exciting  cause,  then,  of  this  outbreak  was  believed  by  Mr 
Power  to  be  a  condition  of  certain  milch  cows  which  had  for  its  outward 
manifestation  an  eruption  on  teats  and  udders,  and  which  was  com- 
municable from  cow  to  cow.  Subcultures  of  the  ulcerous  discharges 
of  the  affected  animals  inoculated  into  calves  produced  a  disease 
having  unmistakable  affinities,  under  some  conditions,  with  the 
disease  in  the  milch  cows,  and  under  other  conditions  with  scarlet 
fever  in  the  human  subject  (Klein).  Now,  it  must  be  added,  that 
scarlet  fever  appeared  simultaneously  in  all  but  one  of  the  five 
localities  to  which  the  milk  was  distributed.  The  exception  received 
none  of  the  milk  from  the  affected  cows  until  later,  when  the  disease 
also  appeared  in  this  district,  owing  to  some  of  the  milk  from  the 
affected  cows  being  sent  there.  When  the  sale  of  the  milk  was  pro- 


216  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

hibited  in  London,  some  of  it  was  clandestinely  distributed  amongst 
the  poor  of  Hendon.  Amongst  those  served,  half  a  dozen  families 
were  invaded  by  scarlatina  at  a  time  when  the  disease  had  ceased  to 
exert  its  influence  in  the  London  districts.  The  intermission  which 
had  occurred  in  the  scarlatina  in  Hampstead  and  St  Pancras  during 
the  ten  days  referred  to  above,  was  at  the  time  when  the  infective 
cows  had  been  moved  into  a  shed  sending  milk  only  to  Marylebone. 
A  few  days  later  they  were  again  moved  into  a  shed  from  which 
milk  was  distributed  to  the  two  former  districts. 

Thus  the  investigation  showed  the  Hendon  farm  to  be  the  main 
source,  and,  as  far  as  could  be  judged,  the  cows  referred  to,  the 
particular  source,  of  the  implicated  milk,  for  the  disease  followed  the 
distribution  of  their  milk.  The  further  inquiry  was  with  regard  to 
the  nature  of  the  disease  or  influence  appertaining  to  these  cows.* 

Sir  George  Buchanan  summarised  for  the  Local  Government 
Board  his  interpretation  of  the  facts,  and  concluded  that  the  Hendon 
disease  was  "a  form  occurring  of  the  very  disease  that  we  call 
scarlatina  when  it  occurs  in  the  human  subject."f  His  views  found 
acceptance  with  a  large  number  of  persons,  but  most  veterinarians 
and  certain  pathologists  were  not  prepared  to  accept  the  matter  as 
proved.  In  consequence,  further  investigations  and  inquiries  were 
instituted,  and  a  controversy  arose.  Sir  George  Brown,  then  head  of 
the  Privy  Council's  Agricultural  Department,  held  (1)  that  the 
Hendon  disease  was  not  confined  to  the  Hendon  farm  from  which  the 
implicated  milk  was  derived,  but  occurred  elsewhere,  and  was  followed 
by  no  scarlet  fever;  (2)  that  the  probable  source  of  infection  at 
Hendon  was  human ;  and  (3)  that  the  alleged  bovine  scarlet  fever 
was  cowpox.\ 

As  a  matter  of  fact,  the  exact  origin  of  the  London  epidemic  at 
that  time  has  not  yet  been,  and  now  probably  never  will  be,  demon- 
strated. Even  at  the  present  time  the  specific  micro-organism  which 
is  the  causal  agent  of  human  scarlet  fever  is  not'  established  or 
proved.  That  is  to  say,  no  micro-organism  has  yet  been  isolated  in 
human  scarlet  fever  which  fulfils  the  postulates  of  Koch.  Much  less 
was  this  the  case  sixteen  years  ago,  when  bacteriological  methods 
were  less  perfect  than  they  are  even  to-day.  From  this  it  follows 
that  the  vera  causa  was  obscure,  and  yet  without  this  link  it  was 
impossible  to  complete  the  chain  of  evidence  by  which  it  could  be 
definitely  known  that  any  disease  of  the  cow  was  responsible  for  the 
epidemic.  The  probabilities  might  be  strong  or  weak,  but  proof  was 

*  Local  Government  Board  Report,  1885,  pp.  73-111  (W.  H.  Power). 

f  Seventeenth  Annual  Report  of  the  Local  Government  Board,  1887-88  (Medical 
Officer's  Supplement),  pp.  13,  14. 

£  For  a  discussion  of  the  whole  subject,  see  Bacteriology  of  Milk  (Swithinbank 
and  Newman),  1903,  pp.  279-304. 


MILK-BORNE  SCARLET  FEVER  217 

wanting.  The  inoculation  experiments,  in  so  far  as  they  yielded 
positive  results,  were  also  open  to  the  same  unreliability.  Unfortu- 
nately, too,  there  was,  on  the  other  hand,  circumstantial  evidence  of 
various  kinds,  which,  while  it  proved  little,  opened  up  a  variety  of 
possibilities  by  which  the  milk  consumed  in  London  might  have 
become  infected.  The  case  was  therefore  unproved.  Nevertheless,  it 
raised  many  important  questions  and  stimulated  much  valuable 
inquiry.  When  milk  becomes  infected  with  scarlet  fever  the  infec- 
tion is  almost  invariably  derived  directly  from  some  person  suffering 
from  the  disease,  recognised  or  unrecognised. 

Scarlet  Fever,  in  not  a  few  milk-epidemics,  has  shown  certain 
modifications  of  a  more  or  less  marked  character.  The  disease  is 
generally  mild,  and  simultaneously  with  an  outbreak  of  the  specific 
disease  due  to  milk,  there  will  not  infrequently  be  found  a  large 
number  of  "  ordinary  sore  throats."  Even  in  the  scarlatinal  cases, 
the  disease  has  a  tendency  to  remain  localised  to  the  throat  (Power). 
The  rash  may  be  evanescent,  and  the  desquamation  is  scanty 
(Parsons).  There  is  also  a  marked  absence  of  post-scarlatinal 
nephritis  or  any  other  kidney  complication  (Parsons,  Buchanan,  and 
others).  A  characteristic  which  has  been  frequently  noted,  and  is 
readily  to  be  understood,  is  the  frequency  of  vomiting  and  diarrhoea, 
rather  particularly  at  the  commencement  of  the  disease  (the  Fallow- 
field  epidemic,  1879,  is  an  illustration).  It  is  probable  that  these  signs 
of  alimentary  irritation  or  poisoning  are  due  to  poisonous  organismal 
products  contained  in  the  milk.  On  more  than  one  occasion  they 
have  led  to  an  appearance  of  intoxication  rather  than  infection. 
Finally,  there  is  a  clinical  feature,  to  which  reference  has  already 
been  made,  and  which  may  bear  a  significance  not  at  first  appreciated, 
namely,  the  comparative  indisposition  of  the  disease  to  spread  by 
contagion.  This  may  be  attributable  to  the  mildness  of  the  disease, 
to  the  small  amount  of  skin  eruption  and  desquamation  commonly 
present,  and  possibly  to  the  fact  that  the  poisonous  properties  of  the 
milk  are  to  a  certain  extent  eliminated  from  the  system  by  the 
vomiting  and  purging.  Every  clinical  sign  which  has  been  noted 
leads  to  the  conclusion  that  the  disease  as  conveyed  by  milk  is 
frequently  mild,  and  therefore  has  both  a  small  mortality,  and  no 
tendency  to  spread  by  contact.  There  is  one  other  point  deserving 
of  mention.  Sir  George  Buchanan  noticed,  in  a  scarlet  fever 
epidemic  with  which  he  had  to  deal,  that  in  persons  who  had  had 
scarlet  fever  at  some  previous  time,  and  who  drank  the  implicated 
milk,  almost  the  only  symptom  of  ill-health  which  they  presented 
was  a  sore  throat.  There  was  no  rash,  no  vomiting,  no  pyrexia, 
although  other  members  of  the  family  under  precisely  similar  circum- 
stances suffered  from  typical  scarlet  fever.  Many  other  workers  have 
confirmed  the  occurrence  of  aberrant  forms  of  milk-borne  scarlatina. 


218  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

Characteristics  of  Milk-borne  Epidemics 

The  following  are  the  chief  characteristics  of  infectious  disease 
carried  by  milk: — 

(a)  There  is  a  special  incidence  of  disease  upon  the  track  of  the 
implicated  milk  supply.     It  is  localised  to  such  areas. 

(b)  Better-class  houses  and  persons  generally  suffer  most. 

(c)  Milk  drinkers  are  chiefly  affected,  and  those  suffer  most  who 
are  large  consumers  of  raw  milk. 

(d)  Women  and  children  suffer  most,  and  frequently  adults  suffer 
proportionately  more  than  children. 

(e)  Incubation  periods  are  shortened. 

(/)  There  is  a  sudden  onset  and  a  rapid  decline. 
(g)  Multiple  cases  in  one  house  occur  simultaneously. 
(h)  Clinically,  the  attacks  of  disease  are  often  mild,  contact  infec- 
tivity  is  reduced,  and  the  mortality  rate  is  lower  than  usual. 

Other  Diseases  Conveyed  by  Milk 

In  addition  to  the  above,  there  are  other  diseases  spread  by 
means  of  polluted  milk.  From  time  to  time  exceptional  cases  have 
occurred  in  which  diseases  like  anthrax,  or  some  forms  of  foot-and- 
mouth  disease  have  been  spread  by  this  means.  But  it  is  not  to 
such  rare  cases  that  we  refer.  There  are  three  very  common  diseases 
in  which  milk  has  been  proved  to  play  a  not  inconsiderable  part, 
viz.,  thrush,  sore  throat,  and  diarrhoea. 

Thrush. — The  mould  which  gives  rise  to  the  curd-like  patches  in 
the  throats  of  children,  and  which  is  known  as  Oidium  albicans, 
frequently  occurs  in  milk.  Soft,  white  specks  are  seen  on  the 
tongue  and  mucous  membrane  of  the  cheeks  and  lips,  looking  not 
unlike  particles  of  milk  curd.  If  a  scraping  be  placed  upon  a  glass 
slide  with  a  drop  of  glycerine,  and  examined  by  means  of  the  micro- 
scope, the  spores  and  mycelial  threads  of  this  mould  will  be  seen. 
The  spores  are  oval,  and  possess  a  definite  capsule.  The  threads  are 
branched  and  jointed  at  somewhat  long  intervals.  Milk  affords  an 
excellent  medium  for  the  growth  of  this  parasite.  Thus  undoubtedly 
we  must  hold  milk  partly  responsible  for  spreading  this  complaint. 
Penicillium,  Aspergillus,  and  Mucor  are  also  frequent  moulds  in 
milk. 

Sore -Throat  Illnesses.— The  obscure  milk-borne  epidemics  of 
which  sore  throat  has  been  the  chief  symptom,  are  among  the 
most  interesting  of  all  the  diseases  conveyed  by  milk.  The  usual 
symptoms  are  congestion  of  the  tonsils  and  mucous  membrane  of 
the  throat,  with  sometimes  ulceration,  enlargement  of  the  cervical 
glands,  and  some  pyrexia,  and  general  malaise.  In  not  a  few 


MILK-BORNE  SORE  THROAT  219 

instances  there  has  been  observed  various  kinds  of  rash  which 
have  generally  been  of  an  evanescent  character.  Where  the  throat 
illnesses  have  occurred  contemporaneously  with  outbreaks  of  scarlet 
fever  or  diphtheria,  it  is  not  unlikely  that  the  condition  was  in  reality 
scarlet  fever  or  diphtheria.  In  South  Kensington,  in  1875,  there 
was  an  outbreak  of  disease  which  attracted  much  attention  at  the 
time,  and  was  officially  investigated.*  The  illness  was  traced  to 
some  cream  consumed  at  a  dinner  party,  and  in  all  twenty  persons 
suffered,  some  of  whom  had  scarlet  fever,  and  others  only  sore 
throat.  But  the  investigation  showed  that  in  the  district  from 
which  the  cream  was  obtained  119  cases  of  sore  throat  had  occurred. 
Dr  Darbishire  described  an  outbreak  of  18  cases  of  sore  throat  at 
Oxford  in  1882,  the  condition  being  characterised  by  marked  severity 
of  throat  symptoms  and  a  disproportionate  amount  of  constitutional 
symptoms.f 

Similar  outbreaks  occurred  in  1881  at  Aberdeen  (300  persons 
affected),  and  Eugby  school  (90  boys)  in  three  school-houses  supplied 
by  one  milkman,  who  did  not  supply  any  other  houses  in  the  school. 
But  he  supplied  houses  in  the  town,  of  which  nearly  50  per  cent, 
were  attacked  with  sore  throat.  Inquiry  showed  that  some  of  the 
milk  used  had  been  obtained  from  a  cow  suffering  from  mastitis. j 
A  similar  outbreak  took  place  in  Edinburgh  in  1888,  and  was 
investigated  by  Cotterill  and  Woodhead;  and  another  at  Dover  in 
1884,  where  there  was  a  sudden  and  severe  outbreak  of  sore  throat 
in  a  localised  area  of  good-class  houses,  affecting  205  persons,  who 
all  obtained  milk  from  one  particular  farm.  The  chief  symptoms 
were  local  inflammation  of  the  throat,  enlargement  of  lymphatic 
glands  in  neck,  and  vesicular  eruptions  preceding  and  accompanying 
the  inflammation.  The  dairyman  obtained  his  supply  from  12  cows 
of  his  own,  and  from  three  farms  in  the  country.  On  one  of  these 
latter  apthous  fever  had  broken  out,  and  it  was  from  this  farm  that 
the  dairyman  obtained  his  implicated  milk  and  cream.  Moreover, 
when  the  supply  from  this  farm  was  diverted  temporarily,  it  set  up 
a  simultaneous  second  outbreak  of  sore  throat. §  In  1890  there 
occurred  an  epidemic  of  sore  throat  at  Craigmore,  which  was  referred 
to  milk  infection.  The  number  of  cases  was  80.  The  disease 
manifested  chiefly  in  the  form  of  severe  sore  throat,  but  in  a 
number  of  the  cases  erysipelas  developed  in  addition.  The  milk 
appears  to  have  been  infected  by  two  milkmaids  who  were  suffer- 
ing from  sore  throat.  Those  attacked  most  severely  had  drunk 
most  of  the  implicated  milk.  A  dog  and  cat  which  had  a  good 

*  Report  of  Medical  Officer  of  Local  Government  Board,  1875,  vol.  vii.,  p.  80. 

f  St  Bartholomew's  Hospital  Reports,  vol.  xx. 

J  Brit.  Med.  Jour.,  1881,  vol.  i.,  p.  657;  vol.  ii.,  p.  415. 

§  Practitioner,  1884,  vol.  i.,  p.  467  (Dr  M.  K.  Robertson). 


220  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

deal  of  the  milk  became  very  ill  with  "  severe  inflammation  of  the 
throat."  * 

In  1892  there  was  an  extensive  outbreak  of  sore  throat  in  Upper 
Clapton.  Dr  King  Warry,  describing  the  symptoms  in  the 
Practitioner,  at  the  time  held  that  the  pathological  condition  was 
scarlet  fever  in  a  mild  form.  His  reasons  for  this  view  were  three : 

(a)  scarlet  fever  attacked  one  member  of  a  family,  and  the  sore 
throat  disease  other  members  who  had  previously  had  scarlet  fever ; 

(b)  both  scarlet  fever  and  sore  throat  patients  were  isolated  together, 
but  those  suffering  from  sore  throats  did  not  contract  the  scarlet 
fever ;  and  (c)  some  of  the  patients  suffering  from  sore  throat  had 
at  the  same  time  certain  symptoms  simulating  scarlet  fever,  such 
as  desquamation  of  the  skin,  kidney  disease,  and  rheumatic  symptoms. 
With  this  view  of  the  specificity  of   throat  illness   under   similar 
circumstances  Dr  Parsons  agrees.f     In  the  Upton  and  Macclesfield 
scarlet-fever  outbreak  of  1889,  there  were  40  cases  of  sore  throat 
which   were  apparently  related  to  scarlet  fever  for  the   following 
reasons: — (a)  The  sore  throat  occurred  in  older  persons  in  whom 
rashes  are  less  prone  to  occur,  and  who  had  had  scarlet  fever ;  (b) 
in  some  cases  there  was  skin  desquamation ;  (c)  when  the  children 
suffered  from  scarlet  fever  the  adults  in  the  same  house  suffered 
from  sore  throat;   (d)  two  cases  of  diphtheria  at  the  same  period 
showed  symptoms   simulating  scarlet  fever;   and   (e)   pyrexia  and 
delirium  were  present  in  the  worst  cases. 

Two  comparatively  small  milk-borne  outbreaks  of  "follicular 
tonsillitis"  were  reported  in  1897,  one  in  Anglesey}:  (15  cases),  and 
the  other  at  Surbiton  §  (30  cases).  The  milk  was  bacteriologically 
examined,  and  StapJiylococcus  pyogenes  and  Streptococcus  pyogenes  were 
found,  but  no  B.  diphtheria.  Bacteriological  examinations  of  the 
patients'  throats  yielded  precisely  similar  results.  A  man  whose 
business  it  was  to  milk  the  cows  was  found  to  be  out  of  health,  with 
well-marked  tonsillitis  and  suppurating  whitlows  on  both  hands. 

In  April  and  May  1900  an  outbreak  of  septic  sore  throat  occurred 
in  North  Hackney  affecting  151  persons  in  eighty-eight  households, 
85  per  cent,  of  which  were  supplied  by  one  milkman. 

A  sore-throat  outbreak  at  Brighton  in  November  1901  was 
investigated  by  Dr  Newsholme.  Out  of  a  total  of  29  persons  in  a 
private  girls'  school,  18  were  affected.  The  chief  symptoms  were 
high  temperature,  rapid  pulse,  tonsillitis  with  fibrinous  exudation 
locally  except  on  the  soft  palate.  In  two  cases  there  was  an 
evanescent  rash.  Dr  Newsholme  was  able,  after  minute  inquiry,  to 

*  Glasgow  Med.  Jour.,  1890,  vol.  xxxiv.,  pp.  241-258. 

f  Report  to  Local  Government  Board,  1889. 

\  Brit.  Med.  Jour.,  1897,  vol.  ii.,  p.  339  (Dr  C.  Grey-Edwards  of  Beaumauris). 

§  Annual  Report  of  Medical  Officer  of  Health,  1897  (Dr  Coleman). 


MILK-BORNE  SORE  THROAT  221 

trace  the  cases  at  the  school  to  one  of  their  number,  who  had  come 
into  the  way  of  infection  derived  from  a  milk  supply  contaminated 
by  infectious  disease  in  three  families  connected  with  the  dairy.* 

In  1902  an  outbreak  of  milk-borne  sore  throat  occurred  at 
Lincoln  (199  cases).  Of  the  total  168  or  85  per  cent,  had  consumed 
the  suspected  milk.  The  outbreak  commenced  suddenly,  lasted  a 
few  days,  and  then  suddenly  terminated.  The  majority  of  the 
victims  were  adults  or  persons  over  twelve  years  of  age.  Females 
were  much  more  affected  than  males.  The  symptoms  of  the  disease 
simulated  scarlet  fever.  There  was  marked  sore  throat  and  swelling 
of  the  tonsils,  which  were  in  many  cases  furred.  On  the  third  or 
fourth  day  of  the  disease  there  was  enlargement  of  the  cervical 
glands,  rash  (like  rotheln),  and  fever.  The  commonest  complications 
were  gastritis  and  rheumatism,  but  there  were  a  number  of  irregular 
conditions  and  varieties  of  rash.  The  poison  in  the  milk  seems  to 
have  existed  in  the  highest  degree  in  the  cream,  and  Klein  isolated 
a  yeast  which  he  considers  related  to  a  yeast  known  heretofore  to 
have  been  associated  with  throat  illness  and  thrush.  It  has  been 
suggested  that  some  relationship  may  exist  between  this  yeast  and 
the  spores  of  rusts,  smuts,  and  mushroom  fungi  consumed  by  the 
cows.  The  whole  circumstances  of  the  case  of  this  outbreak  furnish 
one  of  the  most  interesting  modern  chapters  in  milk  epidemiology.-)- 
In  1903  another  outbreak  occurred  (56  cases)  at  Lincoln  of  a  somewhat 
similar  kind.  In  1902  an  outbreak  occurred  at  Bedford  (42  cases) 
consisting  of  sore  throat,  malaise,  headache,  giddiness,  etc.  Here  also 
the  cream  seemed  more  infective  than  the  milk.  Indeed,  in  several 
families  only  those  who  had  taken  the  cream  suffered.  The  incidence 
was  chiefly  upon  young  adults.J 

A  somewhat  similar  outbreak  occurred  in  October  and  November 
1903,  at  Woking,  in  which  persons  were  infected  in  ninety-eight 
different  houses.  The  illness  was  sore  throat,  with  glandular  enlarge- 
ment and  general  symptoms.  Of  the  ninety-eight  households  affected, 
seventy-six  obtained  their  milk  directly  from  a  source  open  to 
criticism.  Dr  Pierce,  the  medical  officer  of  health,  examined  four  cows 
yielding  the  milk,  and  a  bacteriological  examination  was  made  of  the 
milk.  In  the  result  it  was  found  that  two  of  the  cows  suffered  from 
suppurative  mammitis,  and  the  liquid  yielded  by  these  two  cows 
"  consisted  of  the  most  part  of  pus  such  as  would  be  contained  in  an 
abscess."  This  was  the  character  of  the  milk  which  had  been  con- 
sumed by  the  persons  suffering  from  the  illness.  § 

*  Jour,  of  Hygiene,  1902,  vol.  ii.,  pp.  150-169.  Annual  Report  of  Medical  Officer 
of  Health  of  Brighton,  1901. 

t  Report  to  the  Local  Government  Board,  No.  190,  Oct.  1903  (Dr  L.  W.  Darra 
Mair). 

I  Report  of  Medical  Officer  of  Bedfordshire  County  Council,  1902,  pp.  60-62. 

§  Brit.  Med.  Jour.,  1903,  ii.  p.  1492. 


222  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

The  most  recent  sore-throat  outbreak  due  to  consumption  of 
infected  milk  occurred  in  Finchley  in  1904.  Some  500  cases  came 
to  the  knowledge  of  the  medical  officer  of  health  (Prof.  Kenwood) 
of  the  district,  and  another  fifty  occurred  in  the  outlying  neighbour- 
hood. The  incubation  period  was  twenty-four  to  forty-eight  hours, 
followed  by  enlargement  of  submaxillary  glands,  sore  throat,  fever, 
and  general  malaise.  In  a  few  cases  there  was  a  measles-like  eruption 
on  the  lower  limbs.  Professor  Kenwood  formed  the  opinion  that  the 
epidemic  was  due  to  disease  in  the  cows  furnishing  the  milk,  but  no 
specific  organism  was  discovered.*  A  somewhat  similar  outbreak 
occurred  in  the  same  district  in  1894. 

Pus  in  Milk. — It  may  here  be  stated  that  not  infrequently  milk 
contains  pus  cells,  and  there  can  be  little  doubt  that  such  milk 
may  set  up  disease  in  persons  consuming  it. 

Stokes  and  "Wegefarth  made  an  inquiry  into  the  subject  some 
years  ago,  counting  the  number  of  pus  cells  in  the  field  of  the  micro- 
scope in  milk  from  cows  kept  under  various  conditions  of  insanitation. 
Taking  one  pus  cell  in  the  field  as  a  standard,  Stokes  and  Wegefarth 
found  25  per  cent,  of  the  milks  of  country  cows,  kept  under  good 
conditions,  and  88  per  cent,  of  town  cows,  kept  under  bad  con- 
ditions, contained  pus  cells.  Eastes,  who  made  an  examination  of 
186  London  milks,  found  pus  cells  present  in  30  per  cent.,  muco-pus 
in  48'7  per  cent.,  and  streptococci  in  75 '2  per  cent.f  An  in- 
vestigation of  milk  in  St  Pancras  in  1899  yielded  24  per  cent,  of 
samples  containing  pus  cells.  J  Foulerton,  examining  a  series  of 
milks  from  Finsbury  in  1903,  found  pus  and  allied  cells  in  32  per  cent, 
of  them,  staphylococci  in  28  per  cent.,  and  streptococci  in  32  per 
cent.  Forty  per  cent,  of  the  samples  examined  contained  "foreign 
dirt."§  Mucous  threads  are  commonly  found  in  milk  containing  pus. 
Such  threads  probably  consist  of  nucleo-albumin,  and  when  occurring 
with  pus  cells,  the  condition  of  "  muco-pus  "  is  present.  This  is  held 
to  indicate  inflammatory  lesion  of  the  ducts  of  the  udder,  and  not 
abscess  formation  in  the  substance  of  the  gland.  Blood  corpuscles 
are  not  rare  in  milk,  particularly  soon  after  lactation.  The  last  and 
least  important  kind  of  cell  is  that  of  the  epithelium.  Such  scales 
may  be  derived,  either  from  the  hand  of  the  milker  or  from  the 
teats  of  the  udder.  Epithelial  cells  are  large  and  nucleated.  Milk 
containing  many  blood  cells,  mucous  threads,  and  leucocytes,  and 
milk  containing  any  pus  cells,  should  be  looked  upon  as  unfit  for 
human  consumption.  Eastes,  Hoist,  Mven,  Stokes,  Bergey,  Hirsch, 
and  others,  have  drawn  attention  to  the  ill  effects  which  streptococcal 

*  Special  Report  of  Medical  Officer  of  Stoke  Newinyton,  1904. 
t  Brit.  Med.  Jour.,  1899,  vol.  ii.,  p.  1342. 
I  Report  on  Health  of  St  Pancras,  1899,  pp.  61-66  (Dr  Sykes). 
§  Report  on  Milk  Supply  of  Finsbury,  1903,  p.  44. 


MILK-BORNE  DIARRHOEA  223 

milk  has  upon  persons  consuming  it.  In  the  main  these  are  twofold, 
namely,  gastro-intestinal  diseases  and  sore  throats.  The  evidence 
implicating  streptococcal  milk  is  empirical  and  circumstantial,  and 
yet  it  appears  to  be  growing  in  force  and  volume.  On  the  other 
hand,  streptococcus  has  been  found  in  the  fresh  milk  derived  from 
healthy  udders  (Eeed  and  Ward). 

Milk-borne  Cholera 

The  cholera  bacillus  is  unable  to  live  in  an  acid  medium.  Hence 
its  life  in  milk  is  a  limited  one,  and  generally  depends  upon  some 
alkaline  change  in  the  milk.  Heim  found  that  the  organism  of 
cholera  would  live  in  raw  milk  from  one  to  four  days,  depending 
upon  the  temperature.  D.  D.  Cunningham,  from  the  results  of  a 
large  number  of  investigations  in  India,  concludes  that  the  rapidly 
developing  acid  fermentations  normally  or  usually  setting  in,  con- 
nected with  the  rapid  multiplication  of  other  common  bacteria  and 
moulds,  tend  to  arrest  the  multiplication  of  cholera  bacilli,  and 
eventually  to  destroy  their  vitality.  Boiling  milk  appears,  on  the 
contrary,  to  increase  the  suitability  of  the  milk  as  a  nidus  for 
cholera  bacilli,  partly  by  its  germicidal  effect  upon  the  acid-producing 
microbes,  and  partly  that  it  removes  from  the  milk  the  enormous 
numbers  of  common  bacteria,  which  in  raw  milk  cause  such  keen 
competition  that  the  cholera  bacillus  finds  existence  impossible. 

Professor  W.  J.  Simpson,  sometime  the  Medical  Officer  of  Health 
for  Calcutta,  has  placed  on  record  an  interesting  series  of  cholera 
cases  on  board  the  Ardenclutha,  in  the  port  of  Calcutta,  which  arose 
from  drinking  milk  which  had  been  polluted  with  one  quarter  of  its 
volume  of  cholera-infected  water.  This  water  came  from  a  tank 
into  which  some  cholera  dejecta  had  passed.  Of  the  ten  men  who 
drank  the  milk,  four  died,  five  were  severely  ill,  and  one,  who  drank 
but  very  little  of  the  milk,  was  only  slightly  ill.  There  was  no 
illness  whatever  among  those  who  did  not  drink  the  milk.  In  1894, 
a  milk-borne  outbreak  of  cholera  occurred  in  the  Gaya  Gaol,  affecting 
some  twenty-six  persons. 

Milk-borne  Epidemic  Diarrhoea 

In  1892,  Gaffky  recorded  an  instance  in  which  three  men  con- 
nected with  the  Hygienic  Institute  at  Giessen  were  suddenly  taken 
ill.  They  had  chills,  fever,  diarrhoea,  and  general  symptoms.  The 
only  article  of  diet  of  which  they  had  all  partaken  was  milk,  which 
was  traced  to  a  cow  suffering  from  enteritis.  The  milk  of  this  cow  as 
it  left  the  udder  contained  no  bacteria.  But  bacteria  gained  access 
during  the  milking  from  the  dried  particles  of  fsecal  matter  on  the 


224  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

posterior  portion  of  the  udder.  In  these  particles  was  found  a 
bacillus  which  proved  very  pathogenic  for  mice  and  guinea-pigs,  and 
which  corresponded  to  an  organism  isolated  from  the  stools  of  the 
patients.* 

In  1894  an  outbreak  occurred  at  Manchester/]-  characterised  by 
diarrhaea,  sickness,  and  abdominal  pains.  The  cases  numbered  160 
in  forty-seven  houses,  or  just  50  per  cent,  of  the  houses  served  by  one 
and  the  same  milk-seller.  Eaw-milk  drinkers  were  the  chief  sufferers, 
and  those  not  drinking  the  implicated  milk  did  not  suffer.  Dr 
Niven  visited  the  farm  whence  the  milk  came,  and  found  that  it 
was  the  milk  from  the  farm  itself,  and  not  the  added  milk  from  a 
more  distant  farm,  which  supplemented  the  farmer's  stock  that  had 
caused  the  epidemic,  the  home-farm  milk  only  being  sent  into  the 
affected  district.  Near  the  farm  were  40,000  tons  of  privy-midden 
refuse.  Two  streams  ran  near  the  farm,  meeting  below,  one  fouled 
by  the  drainage  of  the  tip,  and  the  other  being  contaminated  with 
sewage  and  with  matter  from  a  tripe-boiling  place.  The  water  used 
for  washing  the  milk -pails  was  tepid,  and  kept  in  a  foul  cistern. 
The  cows  drank  from  a  pool  which  received  the  drainage  from  the 
cowshed  midden.  The  stored  milk  could  be  readily  contaminated 
from  emanations  from  the  cowshed.  Professor  Dele"pine  examined 
the  milk,  and  found  B.  coli  communis  abundantly  present,  and  Dr 
Niven  elicited  the  fact  that  a  cow  affected  with  inflamed  udder 
("  garget ")  had  been  removed  from  the  farm  and  slaughtered.  The 
outbreak  was  attributed  to  milk  in  any  case,  and  to  the  probable 
infection  of  it  by  the  diseased  cow.  But  Delepine  has  pointed  out 
that  it  is  more  probable  that  the  milk  was  contaminated  with  fsecal 
pollution  rather  than  infectious  disease  of  the  cow.| 

In  1895  §  and  1898 1|  three  outbreaks  of  epidemic  diarrhoea 
occurred  amongst  the  patients  at  St  Bartholomew's  Hospital,  London, 
traceable  in  the  first  two  instances  to  milk,  and  in  the  third,  to  rice 
pudding  made  with  milk.lf  On  Sunday  night,  27th  October  1895, 
an  outbreak  of  diarrhoea  affected  59  in-patients,  all  of  whom  had 
recently  taken  milk,  and  from  the  evacuations  the  spores  of  B.  enteri- 
tidis  sporogenes  was  isolated  by  -Klein.  The  patients  suffered  quite 
irrespective  of  whether  or  not  the  milk  had  been  boiled.  Some 
milk  also,  derived  from  the  same  source  as  the  milk  which  had 
caused  the  poisoning,  was  examined  by  Klein,  and  found  to  contain 
the  spores  of  the  same  organism.  On  Sunday,  6th  March  1898,  a 
second  outbreak  of  severe  diarrhoaa  occurred  in  this  hospital,  affect- 

*  Deut.  Med.  Woch.,  vol.  xviil,  p.  14. 

t  Annual  Report  of  Medical  Officer  of  Health  of  Manchester,  1894  (Dr  Niven). 

I  Jour,  of  Hygiene,  1903,  vol.  iii.,  No.  1,  pp.  76,  77. 

§  Report  of  the  Medical  Officer  of  Local  Government  Board,  1895-96,  pp.  197-204. 

II  Ibid.,  1897-98,  p.  235.    ' 

IT  Ibid.,  1898-99,  p.  336.     Lancet,  7th  January  1899. 


MILK-BORNE  DIARRH 


ing  146  patients,  and  there  was  evidence  on  this  occasion  also  that 
the  medium  of  infection  had  been  milk.  On  5th  August  1898,  a 
third  outbreak  affecting  84  patients  and  2  nurses  took  place  at  the 
same  hospital,  the  vehicle  of  infection  in  this  instance  being  some 
rice  pudding  made  with  milk,  also  said  to  contain  an  organism 
similar  or  identical  with  the  B.  enteritidis  sporogenes.  There  can  be 
no  doubt  that  milk  was  the  agent  of  infection  in  each  of  these  three 
outbreaks.  It  was  in  these  outbreaks  that  the  B.  enteritidis  sporogenes 
of  Klein  was  isolated  and  held  to  be  the  specific  organism.  Dr  Klein 
has  produced  evidence  in  behalf  of  this  bacillus  being  the  true  cause 
of  epidemic  diarrhoea.* 

Other  similar  outbreaks  are  on  record  traceable  to  contaminated 
milk.  Nor  is  the  evidence  on  this  subject  derived  only  from  epidemics. 
Newsholme  has  shown  that  of  the  fatal  cases  of  diarrhoea  in  children 
only  9*4  per  cent,  occur  in  children  which  have  been  breast-fed.-)- 
In  Finsbury  20  per  cent.,  in  Kensington  35  per  cent.,  and  in  Croydon 
12  per  cent.,  were  breast-fed.  From  such  figures  it  is  evident  that 
most  of  the  deaths  of  infants  from  diarrhoea  occur  in  children  who 
have  been  hand-fed.  This  fact  is  now  widely  accepted.  In  one  of  his 
official  reports  {  Dr  Hope,  of  Liverpool,  states  that  "  the  method  of 
feeding  plays  a  most  important  part  in  the  causation  of  diarrhoea : 
when  artificial  feeding  becomes  necessary,  the  most  scrupulous 
attention  should  be  paid  to  feeding-bottles."  Careless  feeding,  in 
conjunction  with  a  warm,  dry  summer,  invariably  results  in  a  high 
death-rate  from  this  cause.  These  two  causes  interact  upon  each 
other.  A  warm  temperature  is  a  favourable  temperature  for  the 
growth  of  the  poisonous  micro-organism;  a  dry  season  affords 
ample  opportunity  for  its  conveyance  through  the  air.  Unclean 
feeding-bottles  are  obviously  an  admirable  nidus  for  these  injurious 
bacteria,  for  in  such  a  resting-place  the  three  main  conditions 
necessary  for  bacterial  life  are  well  fulfilled,  viz.,  heat,  moisture,  and 
pabulum.  The  heat  is  supplied  by  the  warm  temperature,  the 
moisture  and  food  by  the  dregs  of  milk  left  in  the  bottle;  and  the 
dry  air  of  summer  assists  in  transit.  It  becomes  clear  that  diarrhoea 
is,  in  part  at  all  events,  due  to  polluted  milk,  polluted  by  dust  or 
flies,  directly  or  indirectly,  at  the  farm  or  in  the  home. 

Delepine  has  urged  that  milk  is  infected  at  the  farm  or  in  transit, 
as  many  of  the  milks  which  he  examined  and  proved  to  be  virulent 
had  not  been  exposed  to  any  influence  attributable  to  a  consumer's 
home,  but  were  in  fact  infective  before  they  reached  the  consumer. § 

*  Reports  of  Medical  Officer  of  Local  Government  Board,  1895-96, 1896-97, 1897-98, 
1898-99. 

f  Report  on  Health  of  Brighton,  1902,  p.  50. 
J  Report  of  Health  of  Liverpool,  1899,  p.  41. 
§  Jour,  of  Hygiene,  1903,  p.  86. 

P 


226  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

He  considers  the  injurious  properties  of  such  milk  is  due  to  ftecal 
pollution  and  the  action  of  B.  coli  (in  particular  those  coliform 
bacilli  which  produce  a  large  amount  of  acid  and  do  not  coagulate 
milk).  Newsholme  considers  such  contamination  may  be  responsible 
for  setting  up  epidemics  of  diarrhoea  occurring  in  connection  with 
a  particular  milk  supply,  as  in  the  analogous  case  of  epidemics  of 
infectious  diseases,  such  as  typhoid.  But  he  holds  that  the  ordinary 
sporadic  cases  of  diarrhoea,  which  carry  off  single  children  in  large 
numbers  in  urban  districts,  are  due  "  chiefly  to  domestic  infection 
of  milk  or  other  foods,  or  to  direct  swallowing  of  infective  dust."* 
Probably,  we  have  a  double  pollution  of  milk  in  actual  practice, 
one  originating  at  the  farm,  one  brought  about  subsequently.  The 
latter  may  be  produced  by  flies,  or  from  manure  heaps  (Waldo),  or 
from  dust  in  roads  and  yards  of  towns  (Eichards),  or  from  the 
generally  filthy  manipulation  of  the  milk  from  the  time  when  it 
becomes  the  property  of  the  milk -seller  to  the  moment  of  con- 
sumption. It  should  not  be  forgotten  in  this  relation  that  stale 
milk  contains  toxic  properties  altogether  apart  from,  and  in  addition 
to,  actual  bacteria.  It  is  possible  that  the  products  of  organismal 
action  have  a  much  greater  effect  in  the  causation  of  diarrhoea  than 
is  generally  supposed,  f 

Preventive   Measures 

It  is  not  possible  in  the  present  state  of  our  knowledge  in  respect 
of  milk  bacteriology  to  lay  down  very  exact  limits  as  to  what  is,  and 
what  is  not,  unsatisfactory  milk.  A  numerical  standard  of  contained 
organisms  is  not  practicable  at  present.  But  we  think,  it  may  be  said, 
that,  in  any  case,  milk  should  not  be  considered  as  marketable  if  it 
contains  (a)  numerous  pus  cells;  (6)  pathogenic  organisms;  or  (c) 
"organisms  of  indication"  of  contamination.  The  presence  of  vast 
numbers  of  bacteria,  such  as  millions  per  cubic  centimetre,  also 
indicates  unclean  manipulation. 

1.  Among  the  preventive  measures  which  these  conditions  indi- 
cate, cleanliness  of  cows,  dairy-hands, '  and  milk-cans  or  other  milk 
vessels,  stands  first  in  importance.     Refrigeration  of  the  milk,  being 
more  easily  obtained  than  cleanliness,  should  be  insisted  upon  without 
delay.     Similar  measures  are  also  needed  with  regard  to  all  things 
or  persons  coming  in  contact  with   the   milk.     Absolute  bacterial 
cleanliness  is  most  difficult  to  obtain,  if  not  practically  impossible. 
Occasional  infection  must,  therefore,  occur. 

2.  To  guard  against  the  effects  of  accidental  fsecal  infection,  milk 
should  be  consumed  fresh,  when  possible.     "When  it  cannot  be  con- 

*  Report  on  Health  of  Brighton,  1902,  p.  50. 

t  For  a  fuller  discussion  of  the  whole  question  of  the  disease-producing  power 
of  milk,  see  Bacteriology  of  Milk,  1903,  pp.  210-391. 


CONTROL  OF  THE  MILK  SUPPLY         227 

sumed  fresh  it  should  be  rnfri<jerated,  i.e.  kept  at  a  temperature 
below  4°  C.,  for  this  inhibits  the  rapid  multiplication  of  bacteria. 
When  milk  cannot  be  used  fresh  or  refrigerated,  it  should  be  sterilised 
by  heat. 

3.  Greater  domestic  and  municipal  cleanliness  is   an   essential 
requirement.      This  must  include  the  cleanly  preparation  of  food, 
and  particularly  the  handling  and  storage  of  milk;   the  cleansing 
of  milk-bottles;   reduction  of  dust   in   houses,  courts,  and   streets, 
and  protection  of  milk  from  dust  in  shops  and  houses ;  impervious 
roads  and  paving;   and  the  substitution  of   wet-cleansing  for  dry 
cleansing,  and  frequent  cleansing  for  infrequent. 

4.  Lastly,  there  is  needed  "a  crusade  against  the  domestic  fly, 
which  is  most  numerous   at   the   seasons   and   in   the  years  when 
epidemic  diarrhoea  is  most   prevalent,  and  probably  plays  a  large 
part  in  spreading  infection "  (Newsholine). 

METHODS  OF  PROTECTING  AND  PURIFYING  MILK 

After  the  consideration  of  the  somewhat  extensive  category  of 
diseases  which  may  be  milk-borne,  it  will  be  desirable  to  make  brief 
reference  to  some  of  the  means  at  our  disposal  for  obtaining  and 
preserving  good,  pure  milk. 

We  considered  at  the  commencement  of  this  chapter  the  most 
frequent  channels  of  contamination.  If  these  be  avoided  or  pre- 
vented, and  if  the  milk  be  derived  from  cows  in  good  health  and 
well  kept,  the  risk  of  infection  is  reduced  to  a  minimum.  The  two 
things  necessary  are  clean,  healthy  cows  and  clean  methods  of  milking 
and  manipulation.  What  the  Danes  can  do,  other  dairy  workers 
can  do.  The  cow  byre,  the  udder,  the  milk  vessel,*  and  the  milkers 
should  each  be  thoroughly  clean.f  But  we  have  seen  that  much, 

*  Probably  the  best  method  of  cleansing  dairy  utensils  is  by  using  steam  or 
boiling  water  and  soda.  The  advantage  of  boiling  water  is  obvious.  The  addition 
of  soda  enhances  its  value,  as  the  soda  unites  with  the  lactic  acid  present,  forming  a 
soluble  lactate  of  soda,  and  also  with  grease,  a  fat  forming  an  easily  soluble  soap. 
Nor  does  it  injure  or  rust  the  metal  with  which  it  comes  into  contact. 

f  It  may  be  well  to  add  in  a  footnote  an  account  of  the  Danish  method  as 
carried  out  in  England,  for  it  illustrates  in  concrete  form  the  practical  way  of 
reducing  pollution  of  milk  to  a  minimum  :— 

The  principles  and  practice  of  the  Copenhagen  Milk  Supply  Company  have 
been  introduced  into  England,  and  are  being  carried  out  by  Mr  C.  W.  Sorensen 
at  the  White  Rose  Dairy,  West  Huntington,  York.  Mr  Sorensen  is  a  nephew  of 
Mr  Busck,  of  the  Copenhagen  Company,  and  has  been  trained  in  the  Danish 
methods.  His  dairy  farm  at  York  is  carried  on  in  a  similar  manner  to  the 
Copenhagen  Company's  work,  with  this  difference,  that  whilst  the  latter  obtain 
their  milk  from  contributory  farms,  Mr  Sorensen  works  his  own  farm,  and  the 
control  and  management  of  the  cows  is  under  his  direct  and  immediate  supervision. 
The  writer  had  an  opportunity  recently  of  visiting  this  dairy  farm  near  York,  and 
a  brief  description  of  the  most  important  points  may  be  added  here. 

1.  The  health  of  the  cows  is  secured  by  a  special  monthly  inspection  by  the 


228  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

if  not  most,  of  the  pollution  of  milk  arises  after  the  milking  process 
and  during  transit  and  storage  preparatory  to  use.  Bacteria  are 
so  ubiquitous  that  to  prevent  the  entrance  of  any  at  all  is  futile. 
It  is,  therefore,  well  to  bear  in  mind  the  extreme  importance  of 
careful  straining  and  immediate  cooling.  Straining  or  screening  milk 
removes  the  grosser  particles  of  dust,  dirt,  hairs,  etc.,  and  these,  it 
will  be  remembered,  are  the  "rafts"  and  "vehicles"  of  bacteria. 
If  they  are  at  once  removed  therefore,  many  bacteria  will  be  removed 
with  them.* 

York  Corporation  Veterinary  Officer,  Mr  William  Fawdington,  M.R.C.V.S.,  who 
has  authority  to  order  the  disposal  of  any  unhealthy  or  even  suspected  animal, 
and  whose  reputation  and  experience  affords  a  guarantee  of  efficiency  in  this 
important  point.  There  are  about  50  cows  in  all,  10  of  which  are  Jerseys.  The 
feeding  of  the  cows  is  scientifically  carried  out.  No  brewers'  grains,  turnip-tops, 
or  other  unsuitable  foods  are  used,  and  especial  care  is  exercised  in  the  selection 
and  feeding  of  the  cows  supplying  "  Table  or  Nursery  Milk,"  so  as  to  maintain  a 
high  standard  of  richness  and  flavour.  To  ensure  an  abundant  supply  of  pure 
water  for  the  cows  to  drink,  as  well  as  for  cleansing  purposes,  the  farm  has  been 
connected  with  the  York  City  Water  Supply,  which  is  provided  in  a  continuous 
trough  at  the  head  of  the  stalls.  The  cleanliness  and  ventilation  of  the  cow-houses 
receives  special  attention,  and  is  in  every  way  excellent. 

2.  While  no  money  has  been  wasted  on  fancy  fittings  (which  make  the  milk  no 
better,  but  simply  increase  the  cost),  the  proprietor's  aim  has  been  to  keep  every- 
thing sweet  and  clean  from  the  cows  themselves  down  to  the  smallest  utensil.     A 
high-pressure  boiler  has  been  put  in  for  sterilising  all  utensils,  cans,  etc.,  with 
steam. 

The  udders  of  the  cows  are  cleansed  before  milking.  The  milkers  are  clothed 
in  over-alls,  and  wash  before,  and  if  necessary,  during  milking.  The  operation  of 
milking  is  carried  out  under  cleanly  conditions  and  with  clean  utensils.  After 
milking  the  milk  is  strained  by  a  "  Ulax"  strainer. 

3.  Immediately  after  the  milk  is  strained,  prompt  and  efficient  cooling  is  obtained 
by  allowing  it  to  flow  in  a  thin  layer  over  a  corrugated  copper  cylinder,  inside  which 
cold  water  and  ice  are  passed,  thus  reducing  the  temperature  in  a  few  seconds  to  a 
point  at  which  germ  life  cannot  develop.     Clean  milk,  so  treated,  needs  no  **  pre- 
servation."    If  kept  in  a  cool  place  it  will  remain  perfectly  sweet  for  several  days, 
even  in  the  hottest  weather.    Therefore,  no  preservation  or  sterilisation  is  necessary. 

4.  The  usual  practice  of  slopping  milk  about  from  one  can  to  another  in  the 
street — exposed  to  contamination  from  clouds  of  dust,  the  not  always  clean  hands, 
or,  in  wet  weather,  the  dripping  garments  of  the  driver — is  one  so  objectionable 
that  only  long  usage  and  the  absence  of  anything  better  has  made  it  tolerated. 
The  ideal  system,  without  doubt,  is  delivery  in  glass  bottles,  filled  and  sealed  at 
the  dairy,  and  placed  straight  on  the  table  without  the  intervention  of  jugs,  basins, 
or  what  not.     Next  comes  delivery  in  cans,  likewise  filled  and  sealed  at  the  dairy. 
After  that  comes  the  system  of  drawing  the  milk  by  tap  from  a  sealed  can,  which, 
however,  is  much  preferable  to  the  plan  of  dipping  into  an  open  can.     The  entire 
system  at  this  dairy  farm  is  so  arranged  as  to  supply  a  clean  whole  milk  from 
healthy  cows  kept  under  hygienic  conditions,  and  protected  from  dust  and  infection. 
This  desirable  object  is  attained  by  (a)  clean  milking,  (b)  straining,  (c)  cooling,  and 
(d)  bottling  promptly,  efficiently,  and  at  the   dairy   farm.     On   the  whole,   Mr 
Sorensen's  methods  appear  to  represent  the  high  tide  of  dairy  farm  work  in  England. 
But  nothing  is  done  by  him  which  could  not  be  done  by  every  dairy  farmer  in  the 
country. 

*  One  of  the  most  satisfactory  strainers  in  the  market  is  that  known  as  the 
"Ulax."  This  apparatus  consists  of  a  metal  sieve  through  which  the  milk  is  first 
passed.  Then  a  finer  double  sieve  with  a  thin  layer  of  sterilised  cotton-wool  placed 
between  the  two  metal  sieves  acts  as  a  secondary  filter  (see  Fig.  23). 


CONTROL  OF  THE  MILK  SUPPLY 


229 


Low  temperatures,  it  is  true,  do  not  easily  destroy  life,  but  they 
have  a  most  beneficial  effect  upon  the  keeping  quality  of  milk.  It 
has  been  suggested  that  at  the  outset  of  the  process  of  cooling, 
currents  of  air,  inimical  to  bacteria,  are  started  in  the  milk.  If, 
however,  the  temperature  be  lowered  sufficiently,  the  contained 
bacteria  become  inactive  and  torpid,  and  eventually  are  unable  to 
multiply  or  produce  their  characteristic  fermentations.  At  about 
50°  F.  (10°  C.)  the  activity  ceases,  and  at  temperatures  of  45°  F. 
(7°  C.)  and  39°  F.  (4°  C.)  organisms  are  practically  deprived  of  their 
injurious  powers.  If  it  happens  that  the  milk  is  to  be  conveyed 
long  distances,  then  even  a  lower  temperature  is  desirable.  The 
most  important  point  with  regard  to  the  cooling  of  milk  is  that  it 
should  take  place  immediately.  Various  kinds  of  apparatus  are 


FIG.  23.-"  Ulax  "  Filter. 


effective  in  accomplishing  this.  Perhaps  those  best  known  are 
Lawrence's  cooler  and  Pfeiffer's  cooler,  the  advantage  of  the  latter 
being  that  during  the  process  the  milk  is  not  exposed  to  the  air. 
It  must  not  be  forgotten  that  cooling  processes  are  not  sterilising 
processes.  They  do  not  necessarily  kill  bacteria;  they  only 
inhibit  activity,  and  under  favourable  circumstances  torpid  pathogenic 
bacteria  may  again  acquire  their  injurious  faculties.  Hence,  during 
the  cooling  of  milk  greater  care  must  be  taken  to  prevent  aerial 
contamination  than  is  necessary  during  the  process  of  sterilising 
milk.  No  cooling  whatever  should  be  attempted  in  the  stable; 
but,  on  the  other  hand,  there  should  be  no  delay.  Climate  makes 
little  or  no  difference  to  the  practical  desirability  of  cooling  milk, 
yet  it  is  obvious  that  less  cooling  will  be  required  in  the  cold  season. 
The  final  treatment  of  milk  has  in  practice  comprised  the  addition 
of  preservatives,  filtration,  and  sterilisation. 


230  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

Preservatives  are  widely  used,  especially  in  town  milks.  They 
do  not,  as  a  rule,  kill  bacteria  in  milk,  but  merely  stifle  them,  and 
prevent  rapid  multiplication  and  increasing  acidity.  They  disguise 
the  true  condition  of  the  milk  in  which  they  exist.  It  is  to  be  feared 
that  their  systematic  addition  to  milk  tends  to  place  a  premium  on 
uncleanly  and  improper  dairying.  There  is  evidence,  also,  to  show 
that  by  a  cumulative  process  preservatives  may  be  injurious  to 
persons  consuming  the  milk.  The  most  commonly  used  antiseptics 
in  milk  are  borax,  formalin,*  carbonate  of  soda,  and  salicylic  acid.f 

Secondly,  it  is  possible  to  remove  in  part  the  pollution  of  milk 
by  filtration.  Filtration  has  been  practised  for  some  time  by  the 
Copenhagen  Dairy  Company,  by  Bolle,  of  Berlin,  and  various  milk 
companies.  The  filters  used  consist  of  large  cylindrical  vessels 
divided  by  horizontal  perforated  diaphragms  into  five  superposed 
compartments,  of  which  the  middle  three  are  filled  with  fine  sand 
of  three  sizes.  At  the  bottom  is  the  coarsest  sand,  and  at  the  top 
the  finest.  The  milk  enters  the  lowest  compartment  by  a  pipe 
under  gravitation  pressure,  and  is  forced  upwards,  and  finally  is  run 
off  into  an  iced  cooler,  and  from  that  into  the  distribution  cans.  By 
this  means  the  number  of  bacteria  are  reduced  to  one-third.  The 
difficulty  of  drying  and  sterilising  enough  sand  to  admit  a  large 
turnover  of  milk  is  a  serious  one.  This,  in  conjunction  with  the 
belief  that  filtration  removes  some  of  the  essential  nutritive 
elements  of  milk,  has  caused  the  process  to  be  but  little  adopted. 
Dr  Seibert  states  that  if  milk  be  filtered  through  half  an  inch  of 
compressed  absorbent  cotton,  seven-eighths  of  the  contained  bacteria 
will  be  removed,  and  a  second  filtration  will  further  reduce  the 
number  to  one-twentieth.  One  quart  of  milk  may  thus  be  filtered 
in  fifteen  minutes. 

*  S.  Rideal  and  A.  G.  R.  Foulerton  conclude  from  a  series  of  experiments  that 
boric  acid  (1-2000)  and  formaldehyde  (1-50,000)  are  effective  preservatives  for  milk 
for  a  period  of  twenty-four  hours,  and  that  these  quantities  have  no  appreciable 
effect  upon  digestion  or  the  digestibility  of  foods  preserved  by  them  (Public  Health, 
1899,  pp.  554-568). 

f  The  Departmental  Committee  on  Preservatives  and  Colouring  Matters  in  Food, 
1901  (Report,  pp.  xxiv.-xxv.)  recommend: — 

1.  That  the  use  of  Formaldehyde  in  food  and  drink  is  absolutely  prohibited,  and 
the  Salicylic  Acid  be  not  used  in  greater  proportion  than  one  grain  per  pint  or 
pound  respectively  for  liquid  or  solid  food,  its  presence  in  all  cases  to  be  declared. 

2.  That  the  use  of  any  preservatives  or  colouring  matter  in  milk  be  made  an 
offence  under  the  Sale  of  Food  and  Drugs  Acts. 

3.  That  Boric  Acid  preservatives  only  be  allowed  in  cream,  the  amount  not  to 
exceed  0'25  per  cent.,  and  be  notified  on  a  label. 

4.  That  Boric  Acid  preservatives  only  be  allowed  in  butter,  the  amount  not  to 
exceed  0'5  per  cent. 

5.  That  chemical  preservatives  be  prohibited  in  all  dietetic  preparations  for  the 
use  of  children  and  invalids. 

6.  That  the  use  of  Copper  Salts  for  "  greening  "  be  prohibited. 

7.  That  a  Court  of  Reference  be  established  to  supervise  the  use  of  preservatives 
and  colouring  matters  in  foods. 


CONTROL  OF  THE  MILK  SUPPLY  231 


Sterilisation  and  Pasteurisation 

Sterilisation  means  the  use  of  heat  at  or  above  boiling-point,  or 
boiling  under  pressure.  This  may  be  applied  in  one  application 
of  one  to  two  hours  at  212°-250°  F.,  or  it  may  be  applied  at  stated 
intervals  at  a  lower  temperature.  The  milk  is  sterilised — that  is  to 
say,  contains  no  living  germs — is  altered  in  chemical  composition, 
and  is  also  boiled  or  "  cooked,"  and  hence  possesses  a  flavour  which 
to  many  people  is  unpalatable. 

Now  such  a  radical  alteration  is  not  necessary  in  order  to  secure 
non-infectious  milk.  The  bacteria  causing  the  diseases  conveyable 
by  milk  succumb  at  much  lower  temperatures  than  the  boiling- 
point.  Advantage  is  taken  of  this  in  the  process  known  as 
pasteurisation.  By  this  method  the  milk  is  heated  to  167-185°  F. 
(75-85°  C.).  Such  a  temperature  kills  harmful  microbes,  because 
75°  C.  is  decidedly  above  their  average  thermal  death-point,  and 
yet  the  physical  changes  in  the  milk  are  practically  nil,  because 
85°  C.  does  not  relatively  approach  the  boiling-point.  There  is  no 
fixed  standard  for  pasteurisation,  except  that  it  must  be  above  the 
thermal  death-point  of  pathogenic  bacteria,  and  yet  below  the 
boiling-point.  As  a  matter  of  fact,  158°  F.  (70°  C.)  will  kill  lactic 
acid  bacteria  as  well  as  most  disease-producing  organisms  found  in 
milk.  If  the  milk  is  kept  at  that  temperature  for  ten  or  fifteen 
minutes,  we  say  it  has  been  "pasteurised."  If  it  has  been  boiled, 
with  or  without  pressure,  for  half  an  hour,  we  say  it  has  been 
"sterilised."  The  only  practical  difference  in  the  result  is  that 
sterilised  milks  have  a  better  keeping  quality  than  pasteurised,  for 
the  simple  reason  that  in  the  latter  some  living  germs  have  been 
unaffected. 

Sterilisation  may,  of  course,  be  carried  out  in  a  variety  of 
modifications  of  the  two  chief  ways  above  named.  When  the 
process  is  to  be  completed  in  one  event  an  autoclave  is  used,  in 
order  to  obtain  increased  pressure  and  a  higher  temperature.  Milk 
so  treated  is  physically  changed  in  greater  degree  than  in  the  slower 
process.  The  slow  or  intermittent  method  is,  of  course,  based  on 
Tyndall's  discovery  that  actively  growing  bacteria  are  more  easily 
killed  than  their  spores.  The  first  sterilisation  kills  the  bacteria, 
but  leaves  their  spores.  By  the  time  of  the  second  application  the 
spores  have  developed  into  bacteria,  which  in  turn  are  killed  before 
they  can  sporulate. 

The  application  of   sterilisation   to   milk   is   now   very   widely 

adopted  by  corporations,  dairy  companies,  etc.     Eecently  the  writer 

has  had  the  opportunity  of  studying  an  excellent  system  in  vogue  in 

Essex,*  and  which  may  be  mentioned  because  it  seems  to  emphasise 

*  J.  Carson,  Crystalbrook  Farm,  Theydon  Bois,  Essex. 


232  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

principles  which  might  be  practised  all  over  England.  Briefly,  it 
may  be  said  that  Mr  Carson's  system  lays  emphasis  on  five  chief 
points : — 

1.  The  cows  used  are  carefully  selected  for  milking  purposes,  are 
regularly  inspected,  and  have  all  been  tested  with  the  tuberculin 
test.      Their  feeding  is  also   kept  under  strict  control,  no  brewers' 
grains  being  used.     In  summer  the  cows  feed  on  grass,  linseed  oil 
cake,  and  a  small  quantity  of  cotton  cake  and  bran ;  in  winter  they 
have  hay,  mangolds,  maize,  germ  meal,  and  linseed  and  cotton  cake. 
The  farm  is  well  kept,  and  maintained  under  strict  sanitary  control, 
the  health  and  cleanliness  of  the  cows  being  the  one  thing  aimed  at. 

2.  The  cows  are  milked  in  the  byre  adjoining  the   sterilising 
plant.      Both   cows   and   cowsheds   are   continually   maintained   in 
cleanliness.    The  udders  are  cleansed  before  milking,  and  it  is  required 
that  milkers  also   shall   be  clean  in   person   and  management   of 
milking. 

3.  Immediately    after  milking,   the   milk  is   removed    into    an 
adjoining    room,   strained    through   a   metal   screen,   and   at   once 
separated   by  an   ordinary    Laval   separator.       This   separation   is 
adopted  for  purposes  of  purification  only.     The   milk   and   cream 
are  again  mixed,  and  poured  by  means  of  a  mechanical  automatic 
bottle-filler  into  bottles. 

4.  The  milk  in  bottles  is  then,  within  a  few  minutes  of  leaving 
the  udder,  placed  in  the  steriliser  and  maintained  at  212°  F.  for 
twenty   minutes.      The  bottles  have  been  previously  sterilised  at 
220°  F.  for  sixty  minutes. 

5.  After  sterilisation  the  milk  is  cooled  to  53°  F.,  and  kept  at 
that  temperature  till  required  for  delivery. 

We  have  examined  this  milk  chemically  and  bacteriologically, 
and  have  found  it  to  be  of  excellent  quality.  It  is  unquestionably 
an  advantage  to  have  milk  which  is  to  be  sterilised  brought  under 
treatment  at  once,  after  milking.  This  cannot  always  be  done,  and 
hence  it  is  the  custom  of  some  dairy  companies  and  institutions  to 
sterilise  milk  on  its  delivery.  But  it  is  of  extreme  importance  to 
avoid  this  if  practicable.  Whatever  treatment  milk  receives,  be  it 
refrigeration  or  sterilisation,  such  treatment  should  be  applied 
immediately  after  the  milk  is  drawn  from  the  udder.  There  are  a 
large  number  of  appliances  and  different  forms  of  apparatus  now  on 
the  market,  having  for  their  object  the  sterilisation  of  milk.  Our 
object  has  not  been  the  recommendation  of  any  apparatus  or  process, 
but  the  principles  underlying  the  efficient  pasteurisation  and  ster- 
ilisation of  milk. 

The  methods  of  pasteurisation  are  continually  being  modified 
and  improved,  especially  in  Germany,  Denmark,  and  America. 
Most  of  the  variations  in  apparatus  may  be  classed  under  two 


CONTROL  OF  THE  MILK  SUPPLY  233 

headings.  There  are,  first,  those  in  which  a  sheet  of  milk  is  allowed 
to  flow  over  a  surface  heated  by  steam  or  hot  water.  This  may  be  a 
flat,  corrugated  surface  or  a  revolving  cylinder.  The  milk  is  then 
passed  into  coolers.  Secondly,  milk  is  pasteurised  by  being  placed 
in  reservoirs  surrounded  by  an  external  shell  containing  hot  water  or 
steam.  Dr  H.  L.  Eussell  *  has  described  one  apparatus  consisting  of 
a  pasteuriser,  a  water  cooler,  and  an  ice  cooler.  The  pasteuriser  is 
heated  by  hot  water  in  the  outside  casement.  To  equalise  rapidly 
the  temperature  of  the  water  and  milk,  a  series  of  agitators  must  be 
used.  These  are  suspended  on  movable  rods,  and  hang  vertically  in 
the  milk  and  water  chambers.  By  this  ingenious  arrangement,  the 
heat  is  diffused  rapidly  throughout  the  whole  mass,  and  as  the 
temperature  of  the  milk  reaches  the  proper  point,  the  steam  is  shut 
off  and  the  heat  of  the  whole  body  of  water  and  milk  will  remain 
constant  for  the  proper  length  of  time.  The  somewhat  difficult 
problem  of  drawing  off  the  pasteurised  milk  from  the  vat  without 
reinfecting  it  by  contact  with  the  air  is  solved  by  placing  a  valve 
inside  the  chamber,  and  by  means  of  a  pipe  leading  the  pasteurised 
milk  directly  and  rapidly  into  the  coolers.  These  are  of  two  kinds, 
which  may  be  used  separately  or  conjointly.  In  one  set  of  cylinders 
there  is  cold  circulating  water,  in  the  other  finely-crushed  ice. 

In  England,  many  methods  (including  a  number  of  patents)  are 
in  common  use  where  milk  is  pasteurised.  For  instance,  at  the 
Hospital  for  Sick  Children  in  Great  Ormond  Street,  which  is  in 
advance  of  other  London  hospitals  in  this  respect,  milk  is  received 
from  a  well-known  metropolitan  dairy  company  in  quantities  of  200 
quarts  daily,  some  being  delivered  in  the  morning,  and  a  smaller 
quantity  in  the  evening.  The  milk  is  derived  from  healthy  cows, 
and  sanitary  cowsheds,  the  farms  being  placed  under  strict  super- 
vision. On  receipt,  the  milk  is  filtered  through  muslin,  by  downward 
and  upward  filtration,  and  passed  directly  into  a  bottle-filling 
machine.  Clean,  stoppered  bottles  are  kept  in  readiness.  When 
filled,  the  bottles  are  placed  in  circles  in  the  cage  at  the  bottom  of 
the  pasteuriser.  Into  the  centre  of  the  apparatus  is  placed  the 
thermometer.  The  lid  is  closed  down  and  clamped,  and  the  steam  is 
admitted  from  below.  The  temperatures  used  are  160°  F.  (or  71°  C.) 
for  twenty  minutes  in  winter,  and  180°  F.  (or  82°  C.)  for  twenty 
minutes  in  summer.  After  the  elapse  of  this  period,  the  lid  is 
removed,  the  stoppers  of  the  bottles  are  fixed  down,  and  hot  water 
is  admitted  into  the  floor  of  the  apparatus.  To  this  hot  water  is 
slowly  added  cold  water,  and  in  about  forty  minutes  the  pasteurised 
milk  has  been  cooled  down,  and  is  ready  for  use  in  the  wards.  The 
apparatus  is  readily  cleansed  after  use,  and  the  various  parts,  includ- 
ing the  bottles,  stoppers,  etc.,  are  cleaned  daily.  A  somewhat 

*  Report  from  Wisconsin  Agricultural  Experiment  Station,  1896. 


234  BACTERIA  IN   MILK  AND  MILK  PRODUCTS 

similar  apparatus  is  in  use  by  a  Health  Association  at  York,*  which 
has  recently  started  (1903)  the  York  Infants'  Milk  Depot,  after  the 
manner  of  the  Liverpool  and  Battersea  system.  The  apparatus  pro- 
vided for  this  work  is  one  of  the  latest  construction.  It  consists  of 
an  ordinary  oval  cylinder  disinfecting  chamber,  having  doors  at  both 
ends.  The  apparatus  is  lagged,  and  with  outside  steel  casing,  pro- 
vided with  a  steam  distributor  inside,  steam  gauge,  safety  valve, 
thermometers,  ready  for  steam  supply  from  boiler.  In  connection 
with  this  apparatus  there  is  also  provided  a  convenient  size  trolley, 
upon  three  wheels,  together  with  a  steel  frame  holding  three  separate 
platforms,  which  can  be  taken  apart  to  suit  bottles  lor  vessels  of 
larger  sizes.  This  frame  is  mounted  also  upon  wheels  running  in 
grooves,  and  channels  are  fitted  inside  steriliser  to  correspond.  The 
steam  rises  around  the  bottles  from  the  bottom  of  the  cylinder.  The 
trolley  is  fitted  for  both  ends,  and  when  duplicated,  a  "  charge  "  can 
be  taken  from  one  end  of  the  apparatus,  and  a  fresh  one  inserted  at 
the  other.  This  apparatus  can  be  used  as  a  steriliser  or  a  pasteuriser. 

Domestic  pasteurisation  can  be  accomplished  readily  by  heating 
the  milk  in  vessels  in  a  water-bath  raised  to  the  required  tempera- 
ture for  half  an  hour,  or  Aymard's  milk  sterilisers  may  be  used. 

Without  entering  into  a  long  discussion  upon  the  various 
pasteurising  methods  adopted,  we  may  summarise  the  chief  essential 
conditions.  It  need  scarcely  be  said  that  the  operation  must  be 
efficiently  conducted,  and  in  such  a  way  as  to  maintain  absolute  con- 
trol over  the  time  and  temperature.  The  apparatus  should  be  simple 
enough  to  be  easily  cleaned  and  sterilised,  and  economical  in  use. 
Arrangements  must  always  be  made  to  protect  the  milk  from  rein- 
fection during  and  after  the  process.  The  entire  preparation  of 
pasteurised  milk  for  market  may  be  summed  up  in  four  items : — 

1.  Pasteurisation  in  heat  reservoir. 

2.  Rapid  cooling  in  water  or  ice  coolers. 

3.  All  cans,  pails,  bottles,  and  other  utensils  to  be  thoroughly 
sterilised  in  steam  before  use. 

4.  The    prepared    milk    to    be  placed  in   sterilised  bottles,  and 
sealed  up. 

The  quality  of  the  milk  to  be  pasteurised  is  an  important  point. 
All  milks  are  not  equally  suited  for  this  purpose,  and  those  contain- 
ing a  large  quantity  of  contamination,  especially  of  spores,  are 
distinctly  unsuitable.  Such  milks,  to  be  purified,  must  be  sterilised. 
Dr  Eussell  has  laid  down  a  standard  test  for  the  degree  of  contamina- 
tion which  may  be  corrected  by  pasteurisation  by  estimating  the 
degree  of  acidity,  a  low  acidity  (e.g.  0'2  per  cent.)  usually  indicating 

*  The  York  Health  and  Housing  Reform  Association,  established  1901. 
Secretary,  Miss  Hutchinson,  63  Gillygate,  York.  Apparatus  by  Wyttenbach :  a 
central  depot  in  Gillygate,  and  branch  depots  elsewhere  in  the  city. 


CONTROL  OF  THE  MILK  SUPPLY 


235 


a  smaller  number  of  spore-bearing  germs  than  that  which  contains  a 
high  percentage  of  acid. 

Lastly,  while  the  heating  process  is,  of  course,  the  essential  feature 
of  efficient  pasteurisation,  it  must  not  be  forgotten  that  rapid  and 
thorough  cooling  is  almost  equally  important.  As  we  have  seen, 
pasteurisation  differs  from  complete  sterilisation  in  that  it  leaves 
behind  a  certain  number  of  microbes  or  their  spores.  Cooling 
inhibits  the  germination  and  growth  of  this  organismal  residue.  If 
after  the  heating  process  the  milk  is  cooled  and  kept  in  a  refrigerator, 
it  will  probably  keep  sweet  from  three  to  six  days,  and  may  do  so 
for  three  weeks. 

Results  of  Pasteurisation. — Before  leaving  this  subject,  we  may 
glance  for  a  moment  at  the  bacterial  results  of  pasteurisation  and 
sterilisation.  The  two  chief  of  these  are  the  enhanced  keeping 
quality  and  the  removal  of  disease-producing  germs.  The  former  is 
due  in  part  to  the  latter,  and  also  to  the  removal  of  the  lactic  acid 
and  other  fermentative  bacteria.  As  a  general  rule,  these  bacteria 
do  not  produce  spores,  and  hence  they  are  easily  annihilated  by 
pasteurisation.  True,  a  number  of  indifferent  bacteria  are  untouched, 
and  also  some  of  the  peptonising  species.  The  cooling  itself  con- 
tributes to  the  increased  keeping  power  of  the  milk,  especially  in 
transit  to  the  consumer. 

Pasteurised  milks  have  the  following  three  economical  and  com- 
mercial advantages  over  sterilised  milks,  namely  (a)  they  are  more 
digestible,  (&)  the  flavour  is  not  altered,  and  (c)  the  fat  and  lact- 
albumen  are  unchanged.  Professor  Hunter  Stewart,  of  Edinburgh, 
compiled  from  a  number  of  experiments  the  following  instructive 
and  comprehensive  table: — 


No.  of 
Experi- 
ments. 

Average  No. 
of  Microbes 
per  c.c.  in 
Milk  before 
Treatment. 

Temperature 
and 
Duration  of 
Pasteurisation 
in  minutes. 

No.  of  Microbes 
per  c.c.  in 
Pasteurised  Milk 
after  24  hours. 

Soluble 
Albumen 
in 
Fresh  Milk 
per  cent. 

Soluble 
Albumen 
in 
Pasteurised 
Milk 
per  cent. 

Taste  of 
Pasteurised  Milk. 

5 

136,262 

10'  60°  C. 

1722  average 

0-423 

0-418 

Unaffected 

4 

53,656 

30'  60°  C. 

1  sterile 

0-435 

0-427 

3  averaged  955 

12 

78,562 

10'  65°  C. 

6  sterile 
6  averaged  68  6 

0-395 

0-362 

Not    appreci- 
ably affected 

12 

132,833 

30'  65°  C. 

9  sterile 

0-395 

0-333 

M 

3  averaged  233 

13 

49,867 

10'  70°  C. 

sterile 

0-422 

0-269 

Slightly  boiled 

9 

38,320 

30'  70°  C. 

0-421 

0-253 

<4 

2 

77,062 

10'  75°  C. 

0-38 

0-07 

Boiled 

3 

48,250 

30'  75°  C. 

0-38 

0-05 

w 

1 

1,107,000 

10'  80°  C. 

0-375 

o-oo 

M 

1 

1,107,000 

30'  80°  C. 

0-375 

o-oo 

" 

236 


BACTERIA  IN  MILK  AND  MILK  PRODUCTS 


It  will  be  admitted  that  this  table  exhibits  much  in  favour  of 
pasteurisation;  yet  the  crucial  test  must  ever  be  the  effect  upon 
pathogenic  bacteria.  Fliigge  has  conducted  a  series  of  experiments 
upon  the  destruction  of  bacteria  in  milk,  and  he  states  that  a 
temperature  of  158°  F.  (70°  C.)  maintained  for  thirty  minutes  will 
kill  the  specific  organisms  of  tubercle,  diphtheria,  typhoid,  and 
cholera.  MacFadyen  and  Hewlett  have  demonstrated,*  by  sudden 
alternate  heating  and  cooling,  that  70°  C.  maintained  for  half  a 
minute  is  generally  sufficient  to  kill  suppurative  organisms,  and  such 
virulent  types  of  pathogenic  bacteria  as  B.  diphtheria,  B.  typhosus, 
and  B.  tuberculosis. 

Respecting  the  numerical  diminution  of  microbes  brought  about 
by  pasteurisation  and  sterilisation  respectively,  we  may  take  the 
following  series  of  experiments.  Dr  H.  L.  Russell  f  tabulates  the 
immediate  results  of  pasteurisation  as  follows : — 


Number  of  Micro-organisms  per  c.c. 

Unpasteurised. 

Pasteurised. 

Minimum. 

Maximum. 

Average. 

| 
Min.    i      Max. 

Av. 

Full  cream 
milk.     . 
Cream  .     . 

25,300 
425,000 

18,827,000 
32,800,000 

3,674,000 
8,700,000 

0          37,500 
0         57,000 

6,140 
24,250 

As  regards  the  later  effect  of  the  process,  he  states  that  in  fifteen 
samples  of  pasteurised  milk  examined  from  November  to  December, 
nine  of  them  revealed  no  organisms,  or  so  few  that  they  might 
almost  be  regarded  as  sterile;  in  those  samples  examined  after 
January,  the  lowest  number  was  100  germs  per  c.c.,  while  the  average 
was  nearly  5000.  With  the  pasteurised  cream  a  similar  condition 
was  to  be  observed.  Other  workers  hold  that  from  95  to  99  per 
cent,  of  all  bacteria  are  removed  by  pasteurisation. 


Summary  of  Practical  Control  of  Milk  Supply 

Briefly,  it  may  be  said  that  the  requirement  is  a  pure  milk  supply, 
that  is : — 

(1)  A  clean,  whole  milk,  unsophisticated  and  without  preserva- 
tion; 

*  Jenner  Institute  of  Preventive  Medicine  (First  Series  Transactions). 
f  Centralblatt  far  Bakteriologie,  etc.,  Abth.  ii. 


SPECIALISED  MILK  237 

(2)  To   be   derived   from   healthy   cows,  guaranteed   free   from 
tuberculosis   by   the   tuberculin   test,  and   living   under   clean   and 
sanitary  conditions; 

(3)  To  be  obtained  by  clean  methods  of  milking,  to  be  strained, 
and  to  be  protected  from  contamination  by  dust  or  dirt,  or  from 
infection  by  disease  of  milker; 

(4)  To  be  kept  cool  by  means  of  refrigeration  from  the  time  it 
leaves  the  cow  to  the  time  it  reaches  the  consumer,  and  not  to  be 
exposed  to  dust  or  uncleanliness  in  any  way  from  the  vessels  in 
which  it  is  placed  or  from  the  persons  by  whom  it  is  handled.* 

Specialised  Milk  Supplies  for  Infants. — The  movement  for  'the  supply  of 
modified  milk  for  the  use  of  infants,  particularly  of  the  artisan  class,  has  now  become 
a  considerable  one,  both  in  Europe  and  America.  Broadly  speaking,  the  systems  repre- 
sented in  England  are  (i.)  the  Municipal  Milk  Depot  (Liverpool,  Battersea,  Bradford, 
etc.),  and  (ii.)  the  Rotch  system  (Walker-Gordon  Laboratories),  (i.)  There  can  be 
little  doubt  that  this  kind  of  milk  supply  may  be  of  great  service  for  the  children  of 
the  poor,  in  the  reduction  of  infantile  mortality  due  to  the  use  of  contaminated  or 
infected  milk,  and  in  special  cases  calling  for  special  treatment.  It  is  not,  however, 
of  the  nature  of  control  of  the  milk  supply,  but  rather,  of  a  specialised  supply,  to  meet 
special  needs.  There  is  evidence  to  show  that  at  Liverpool,  Battersea,  and  other 
places,  it  has  had  beneficial  results  in  this  special  direction.  It  has,  however,  several 
limitations,  unless  properly  managed.  Its  object  being  the  saving  of  life  and  pre- 
vention of  infant  diseases,  it  is  necessary  that  the  system  should  be  individualised. 
Each  mother  must  be  separately  advised,  each  infant  inspected  and  weighed  periodi- 
cally, each  home  supervised,  the  condition  of  the  milk  regularly  tested,  and  the 
source  of  the  milk  kept  under  control,  the  cows  and  cowsheds  from  which  the  milk 
is  derived  being  supervised  by  a  veterinary  surgeon  and  the  Medical  Officer  of 
Health.  And  here,  in  any  event,  the  quality  of  the  milk  used  must  reach  a  high 
standard,  chemically  and  bacteriologically.  If  these  conditions  are  not  fulfilled,  it 
would  appear  that  a  municipal  sterilised  milk  supply  can  only  be  a  palliative  measure 
of  transient  usefulness.  The  chief  desideratum  is  a  naturally  pure  milk  supply,  rather 
than  an  artificially  purified  and  humanised  supply.  The  latter  question  is  one 
certainly  requiring  careful  consideration,  but  of  a  different  nature  to  the  former.  If 
undertaken  by  a  Local  Authority,  it  would  appear  desirable  to  do  it  very  thoroughly, 
after  the  manner  of  Budin  and  Variot  in  Paris,  each  case  being  under  strict  medical 
supervision. 

A  typical  municipal  milk  depot  in  this  country  is  described  in  the  following 
words  : — 

The  milk  is  supplied  by  a  local  dairyman,  and  arrives  in  the  early  morning.  It  is 
guaranteed  free  from  chemical  preservatives,  and  to  contain  not  less  than  3*25  per 
cent,  butter  fat,  and  8 '75  per  cent,  of  solids  not  fat,  and  cream  which  must  contain  not 
less  than  50  per  cent,  of  butter  fat.  The  milk  must  be  drawn  from  healthy  cows, 
stabled  and  milked  under  clean  and  sanitary  conditions.  Utensils,  etc.,  used  must 
be  thoroughly  clean.  These  and  other  requirements  are  set  out  in  the  contract.  The 
first  process  is  the  modification  or  humanisation.  Three  modifications  are  employed. 
The  first  contains  one  part  milk  to  two  of  water,  seven  ounces  of  cream  and  seven  of 
lactose  being  added  to  each  gallon  of  the  mixture.  This  modification  is  given  to 
infants  under  three  months  old.  The  second  modification,  which  is  given  to  infants 
between  three  and  six  months  old,  consists  of  equal  parts  of  milk  and  water,  with 
five  ounces  of  cream  and  lactose  added  per  gallon.  The  third  consists  of  two  parts 

*  For  details  respecting  control  of  milk  supply,  see  Bacteriology  of  Milk,  pp. 
452-599 ;  also,  Report  on  Health  of  City  of  Manchester,  1902  ;  Report  on  Milk  Supply 
of  Finsbury,  1903;  The  Milk  Supply  0/200  Cities  and  Toivns  (U.S.A.  Depart,  of 
Aaric.,  1903,  Bulletin  46);  Brit.  Med.  Jour.,  1904,  vol.  ii.,  pp.  421-429  (Newman). 


238 


BACTERIA  IN  MILK  AND  MILK  PRODUCTS 


milk  to  one  of  water,  with  three  ounces  of  cream  and  lactose  added  per  gallon,  and 
is  given  to  infants  over  six  months  old.  * 

The  milk  having  been  modified,  it  is  bottled,  and  the  number  of  bottles  and  the 
quantities  contained  are  set  out  below  : — 


Xo. 

Age  of  Infant. 

No.  of 
Bottles 
per  day. 

Amount 
per 
Bottle. 

Amount 

c 

1 

Below  2  weeks  old     . 

9 

11  oz. 

13i  oz. 

2 

Between  2  weeks  and  2  months  old 

9 

2.V 

22£ 

3 

2  and  3  months  old 

8 

3 

24 

4 

3  and  4  months  old 

7 

4 

28 

5 

4  and  5  months  old 

7 

4.V 

3H 

6 

5  and  6  months  old 

7 

5 

35 

7 

6  and  8  months  old 

6 

6 

36 

8 

8  and  12  months  old    . 

6 

7 

42 

After  bottling,  the  milk  is  heated  by  steam  under  pressure  for  some  20-30  minutes, 
and  kept  at  a  temperature  of  212°  F.  for  from  five  to  ten  minutes.  It  is  allowed  to 
cool,  and  then  is  supplied  to  the  consumers.  Each  mother,  on  first  coming  to  the 
depot,  is  given  a  leaflet  of  instructions  as  to  the  proper  method  of  using  the  milk. 
The  method  is  very  simple.  When  feeding  time  arrives,  all  she  has  to  do  is  to  place 
the  bottle,  unopened,  in  some  warm  water  till  the  milk  has  reached  body  tempera- 
ture. The  bottle  is  then  opened,  a  small  teat  put  on  the  mouth  of  the  bottle,  and 
the  baby  takes  its  milk  from  the  sterilised  bottle  direct.  The  use  of  the  long-tube 
feeding-bottle  is  obviated. 

This  process  of  humanisation  adapts  the  milk  to  the  infant's  digestive  organs,  the 
sterilisation  kills  the  germs,  and  as  the  bottle  is  not  opened— or  should  not  be  opened 
from  the  time  it  enters  the  steriliser  until  the  infant  is  ready  to  take  milk  from  it 
direct  (no  feeding-bottle  should  be  used)— home  contamination,  unless  it  is  wilful  or 
due  to  extreme  carelessness,  is  prevented. 

It  may  be  convenient  briefly  to  summarise  the  chief  advantages  and  disadvantages 
of  this  system  : — Advantages.— (I)  Infants  fed  on  depot  milk  receive  a  modified  milk 
suited  to  their  digestive  capacities.  (2)  The  milk  is  free  from  injurious  and  other 
bacteria.  (3)  The  mother  is  saved  labour,  and  mistakes  are  prevented,  as  the 
preparation  of  food  for  infants,  etc. ,  is  not  necessary  if  modified  and  sterilised  milk 
is  obtained  ready  prepared.  (4)  Home  contamination  of  milk  is  avoided.  These 
four  advantages  are,  in  my  judgment,  of  great  value.  Disadvantages. — (1)  The  object 
being  the  saving  of  life  and  the  prevention  of  infant  disease,  it  is  necessary  that  such 
a  system  should  be  individualised  and  placed  under  direct  medical  supervision. 
Indiscriminate  use  of  such  milk  is  undesirable,  and  the  hand-feeding  of  infants 
requires  so  much  intelligent  care  that  it  should  not  be  generally  recommended. 
(2)  There  must  inevitably  result  from  any  success  of  this  system  a  tendency  or  risk 
for  mothers  to  feed  their  infants  on  such  milk,  instead  of  nursing  them  in  the  natural 
way.  Therefore,  such  milk  should  only  be  provided  for  those  infants  which  for  some 
adequate  reason  cannot  be  nurtured  on  human  milk.  Such  infants  are  the  exception 
and  not  the  rule,  and  it  is  undesirable  to  adopt  any  method  which  tends  to  lessen 
maternal  feeding  or  maternal  responsibility.  There  are  cases,  and  these  not  a  few, 
where  depot  milk  solves  the  problem  of  infant  feeding,  especially  in  large  towns.  But 
anything  which  tends  in  the  direction  of  placing  the  responsibility  of  rearing  infants 
on  the  municipality  is  to  be  deprecated.  It  is  not  a  question  of  sentiment,  but  of 
fact,  to  say  that  the  great  need  of  the  present  time  in  respect  of  this  infant  problem 
is  a  better  home  life,  more  maternal  care  and  feeding  rather  than  less,  and  a  more 


*  These  quantities  are  of  course  dependent  on  the  proportions  of  fat,  sugar,  and 
albuminoid  substances  originally  present  in  the  milk  dealt  with. 


SPECIALISED  MILK  239 

intelligent,  cleanly,  and  simple  mode  of  rearing  infants.  Infants  should  be  breast-fed, 
and  anything  which  relieves  the  healthy  mother  from  this  duty  should  not  be  looked 
upon  favourably  until  it  has  absolutely  justified  its  worth.  (3)  The  cost  is  at  present 
in  many  places  prohibitive.  (4)  The  evidence  of  benefit  is  not  yet  of  a  conclusive 
or  sufficient  nature  to  form  an  opinion  as  to  how  far  the  use  of  depot  milk  reduces  the 
infant  death-rate.  But  there  can  be  no  doubt  that,  indirectly,  benefit  is  derived. 

Infantile  mortality  has  a  definite  relationship  to  (a)  the  feeding  of  infants  ;  (b) 
personal  care  of  infants  by  parents  ;  (c)  housing  accommodation ;  and  (d)  certain 
meteorological  conditions  affecting  temperature  and  the  dissemination  of  dust.  Other 
elements  enter  into  the  problem,  but,  so  far  as  municipal  action  is  concerned,  those 
are  the  four  main  elements.  If  we  can  succeed  in  raising  the  quality,  as  regards 
purity,  of  the  milk  on  which  infants  are  fed,  we  shall  at  the  same  time  educate  and 
improve  the  sense  of  duty  towards  their  infants  on  the  part  of  parents.  The  mischief 
lies  in  polluted  milk.  The  sources  of  the  pollution  are  not  only  in  unsatisfactory 
methods  of  milking,  and  in  storing  and  conveying  the  milk  supplied,  but  also  in 
dirty  domestic  conditions,  and  particularly  in  carelessness  in  the  use  of  feeding- 
bottles.  Successfully  to  attack,  by  municipal  administration,  all  the  sources  of 
pollution,  is  at  present  impossible,  but  the  ideal  of  public  health  administration  in 
respect  of  infant  feeding  is  a  pure  milk  supply  which  needs  no  sterilisation ;  and 
towards  that  end  all  our  efforts  should  be  directed.  Modification  of  such  cow's  milk 
for  infant  use  will  still  be  necessary.  Meanwhile  we  must  do  the  best  possible 
under  existing  conditions,  and  that  involves  sterilisation,  modification,  and  protection 
from  home  contamination.  It  is  also  essential  that  such  a  milk  supply  should  be 
under  medical  supervision,  and  adopted  only  in  suitable  cases.  Without  entering 
into  unnecessary  details,  it  would  appear  that  there  are  five  possible  means  of 
supplying  such  a  suitable  and  pure  milk  for  infants  :— (1)  By  means  of  municipal  milk 
depots  (vide  supra) ;  (2)  by  one  or  more  milk-vendors  or  dairymen  undertaking,  by 
private  enterprise,  to  furnish  such  modified  milk  (certified)  under  medical  super- 
vision ;  (3)  by  obtaining  such  a  supply  from  some  central  institution,  company,  or 
society,  such,  for  example,  as  the  Walker-Gordon  Laboratory ;  (4)  by  means  of 
medical  milk  commissions,  as  is  done,  in  part,  by  the  milk  commissions  established 
in  the  United  States  of  America ;  and  (5)  by  means  of  a  voluntary  health  society 
supplying  such  milk  under  necessary  supervision  and  control  of  sources  and  usage, 
as  is  done,  in  part,  by  the  York  Health  and  Housing  Association. 

The  essential  points  requiring  attention  are  such  modification  of  the  cow's  milk 
as  will  make  it,  like  human  milk,  suitable  for  infant  consumption,  absolute  control 
of  its  source  and  handling  prior  to  its  modification,  and  prevention  of  home  contami- 
nation by  delivery  in  sealed  bottles.  At  present  it  would  also  be  necessary  to 
pasteurise  or  sterilise  such  milk. 

(ii. )  The  Rotch  system  was  introduced  by  Dr  Thomas  Morgan  Rotch  of  Harvard 
University,  and  is  now  in  operation  in  some  eighteen  or  twenty  cities  in  the  United 
States  and  Canada,  and  has  also  a  centre  in  London.  The  system,  which  has  for 
its  object  the  betterment  of  infant  feeding,  consists  in  controlling  the  milk  supply  by 
controlling  the  farms,  and  establishing  a  chain  of  protection  from  the  time  the  milk 
leaves  the  cow  until  it  arrives  at  the  mouth  of  the  infant.  But  in  addition  to  this 
scheme  of  protection,  there  is  also  combined  with  it  a  scheme  of  modification  of  the 
milk  to  make  it  meet  more  exactly  the  requirements  of  infant  feeding.  The  two- 
fold function  of  the  Rotch  system  may  be  briefly  referred  to  :— (a)  Protection  of  the 
Milk. — With  this  object  in  view  Rotch  made  a  number  of  recommendations  similar 
to  those  laid  down  by  various  Milk  Commissions,  which  latter  indeed  took  many  of 
Rotch's  proposals  for  their  model.  At  the  farms  supplying  milk  under  this  system, 
the  breed  of  the  cow  and  its  food  are  matters  which  receive  primary  attention.  In 
America  the  Holstein  has  been  found  to  be  the  best  for  its  adaptability  for  infant 
feeding.  The  cow  itself  must  be  regularly  and  wisely  fed  on  the  basis  to  which 
reference  has  been  made.  There  is  regular  grooming  and  good  housing.  The  cow- 
house has  cemented  walls,  ceilings,  and  floors,  and  is  properly  drained  and 
frequently  cleansed.  A  most  careful  supervision  of  the  cow's  health  is  maintained, 
and  if  in  any  way  abnormal,  the  cow  is  isolated  until  in  normal  health.  Careful 
tuberculin  testing  is  made  of  each  cow  used,  and  the  milk  of  each  cow  also  under- 
goes microscopical  examination  for  the  purposes  of  detecting  pus  cells,  colostrum 


240  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

cells,  bacteria,  etc.  The  milkers  are  under  strict  medical  supervision,  and  regula- 
tions are  enforced  in  respect  of  "  cleanliness."  Cows  are  milked  in  their  own  stalls, 
but  immediately  after  milking  the  milk  is  taken  in  closely-covered  vessels  to  the 
milk-room,  where  it  is  cooled  and  screened.  The  milk-room  is  a  specially  prepared 
chamber,  having  smooth  surfaces  of  polished  cement,  and  specially  constructed 
ventilators  with  cold-water  sprays  to  moisten  the  air  and  prevent  dust  gaining 
access,  asepsis  being  the  requirement.  The  milk  is  now  ready  for  the  laboratory. 
(6)  Modification. — The  object  of  the  Walker-Gordon  Laboratories  is  first  to  insure 
and  distribute  the  naturally  pure  milk ;  and  secondly,  to  provide  a  place  where 
different  combinations  of  milk  may  be  put  up,  according  to  the  prescriptions  of 
medical  men,  with  accuracy,  and  under  such  conditions  of  cleanliness  and  asepsis  as 
to  insure  the  best  possible  food  for  infant  feeding.  The  necessity  for  modification 
arises  from  two  facts,  namely,  that  milk  varies  in  constituent  percentages,  and  to 
obtain  a  regular  and  uniform  constitution,  modification  is  necessary  ;  and  secondly, 
some  children  require,  for  one  reason  or  another,  a  milk  containing  certain  per- 
centages of  the  various  constituents.  Thus  the  patient  can  receive  on  the  physician's 
order  a  mixture  of  the  percentages  called  for,  made  up  of  either  separated  cream  or 
gravity  cream,  separated  milk  or  whole  milk.  Twelve  years'  experience  in  Boston, 
U.S.A.,  seem  to  indicate  the  practicability  of  this  system  in  preventing  the  summer 
diarrhoea  of  infants  due  to  contaminated  milk. 

In  addition  to  these  two  types,  there  are  various  similar  methods  in  vogue,  each 
of  which  has  its  points  of  advantage.* 

BACTERIA  IN  MILK  PKODUCTS 

Cream  is  generally  richer  in  bacteria  than  milk.  Set  cream 
contains  more  bacteria  than  separated  cream,  but  germs  are  abundant 
in  both.  The  number  of  organisms  found  in  cream  is  enormous. 
Probably  no  other  natural  medium  contains  more.  We  have 
frequently  examined  fresh  cream  in  the  country,  and  found  it  to 
contain  more  than  100,000,000  bacteria  per  c.c.  It  is  not  only  a 
favourable  medium.  It  is  the  filter,  so  to  speak,  of  milk.  For,  as 
the  cream  rises,  the  milk  parts  with  more  than  90  per  cent,  of  its 
contained  bacteria.  Conn  and  Estenf  found  110,000,000  of  bacteria 
per  c.c.  in  unripened  cream  (average  of  four  examinations)  and 
284,000,000  in  the  same  cream  ripened  (average  of  four  examinations). 
Cream  obtained  from  a  creamery  gave  an  average  on  eight  examina- 
tions of  56,000,000  organisms  per  c.c.  unripened,  and  350,000,000 
organisms  per  c.c.  ripened.  Other  examples  of  unripened  cream 
averaged  more  than  90,000,000,  but  when  ripened  averaged  over 
300,000,000.  Normally  ripened  cream  probably  averages  four  or 
five  hundred  millions  of  bacteria  per  c.c.,  which  is  greatly  in  excess 
of  any  other  natural  media.  The  number  of  organisms  in  unripened 
cream  varies  widely. 

The  most  characteristic  feature  of  cream-ripening  is  the  growth  of 
the  acid-producing  organisms,  chiefly  B.  acidi  lactici,  and  the  decline 
of  the  liquefying  and  extraneous  organisms.  B.  acidi  lactici  is  found 
in  very  small  numbers  in  fresh  milk,  as  we  have  already  pointed  out, 

*  For  full  account,  see  Jour,  of  Hygiene,  1904,  p.  329  (McCleary). 
t  Thirteenth  Annual  Report  of  the  Storr's  Agricultural  Expt.  Sta.,  Connecticut, 
1900,  pp.  13-33. 


BUTTER  AND  CHEESE  241 

and  also  in  cream.  But  as  the  ripening  process  proceeds  with 
uniform  regularity,  the  numbers  of  this  organism  rapidly  increase. 
Kollin  Burr  considers  these  lactic  bacteria  gain  access  to  the  milk 
from  outside  sources.*  Buttermilk  and  whey  vary  much  in  their 
bacterial  content. 

Butter  necessarily  follows  the  standard  of  the  cream.  But,  as 
the  butter  fat  is  not  well  adapted  for  bacterial  food,  the  number  of 
bacteria  in  butter  is  usually  less  than  in  cream.  Butter,  when  first 
made,  may  contain  many  million  bacteria  per  gramme.  After  a 
few  days  only  two  or  three  million  may  be  found,  and  if  butter 
is  examined  after  it  is  several  months  old,  it  is  often  found  to  be 
almost  free  from  germs;  yet  in  the  intervening  period  a  variety 
of  conditions  are  set  up  directly  or  indirectly  through  bacterial 
action.-)-  Rancid  butter  is  largely  due  to  organisms.  Putrid  butter 
is  caused,  according  to  Jensen,  by  various  putrefactive  bacteria,  one 
form  of  which  is  named  Bacillus  fcetidus  lactis.  This  organism  is 
killed  at  a  comparatively  low  temperature,  and  is,  therefore,  com- 
pletely removed  by  pasteurisation.  Hi-flavoured  butter  may  be  due 
to  germs  or  an  unsuitable  diet  of  the  cow  and  a  retention  of  the  bad 
quality  of  the  resulting  milk.  Lardy  and  oily  butters  have  been 
investigated  by  S torch  and  Jensen,  and  traced  to  bacteria.  Lastly, 
litter  butter  occasionally  occurs,  and  is  due  to  fermentative  changes 
in  the  milk,  or  the  presence  of  acid-producing  organisms  in  the 
butter  such  as  B.  fluorescens  liquefaciens,  Oidium  lactis,  and  Clado- 
sporium  butyri  (Jensen).  Butter  may  also  contain  pathogenic 
bacteria,  like  tubercle.  The  B.  coli  can  live  for  a  month  in  butter. 

Cheese  suffers  from  very  much  the  same  kind  of  "diseases"  as 
butter,  except  that  chromogenic  conditions  occur  more  frequently. 
Most  of  the  troubles  in  cheese  originate  in  the  milk.J  The  number 
of  bacteria  in  cheese  is  naturally  less  than  that  present  in  milk  or 
cream.  The  closer  texture  and  consistence  of  cheese,  coupled  with 
the  lessened  degree  of  moisture,  are  sufficient  factors  to  account  for 
this.  Nevertheless,  cheese  contains  a  considerable  number  of  organ- 
isms. Adametz  found  that  freshly  precipitated  curd,  moulded  in 
the  press  and  freed  from  excess  of  whey,  contained  between  90,000 
and  140,000  micro-organisms  per  gramme,  a  comparatively  large 
number  of  them  having  the  power  of  liquefying  gelatine,  or,  in 
other  words,  they  possessed  a  peptonising  ferment.  During  the 
period  of  ripening,  the  bacterial  content  of  the  cheese  gradually 
rose  to  850,000  in  Emmenthaler  cheese,  and  5,600,000  per  gramme, 

*  Thirteenth  Annual  Report  of  Storr's  Agricultural  Expt.  Sta.,  Connecticut, 
1900,  pp.  66-81;  also  Centralb.  f.  Bakt.,  Abth.  ii.,  1902,  p.  236. 

t  Keeping  Quality  of  Butter  (L.  A.  Rogers),  U.S.  Dep.  of  Agriculture,  1904, 
Bull.  57. 

J  Board  of  Agriculture  Report  on  Cheddar  Cheese  Making  (F.  J.  Lloyd),  1899, 
pp.  78,  103. 

Q 


242  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

in  a  soft  household  cheese.  Only  a  small  percentage  of  these  are 
of  the  peptonising  species.  Tides  of  organisms  occur  in  cheese,  as 
in  butter  and  milk. 

Method  of  Examination  of  Butter  and  Cheese. — Several  grains  of 
the  butter  or  cheese  should  be  placed  in  a  large  test-tube,  which  is 
then  two-thirds  filled  with  sterilised  water  and  placed  in  a  water- 
bath  at  about  45°  C.  until  the  butter  or  cheese  is  melted  or  "  washed." 
A  small  quantity  may  then  be  added  to  gelatine  or  agar  and  plated 
out  on  Petri  dishes,  or  in  flat-bottomed  flasks,  in  the  usual  way. 
After  which  the  tube  may  be  well  shaken  and  returned  to  the  bath 
inverted.  In  the  space  of  twenty  or  thirty  minutes  the  butter  or 
cheese  has  separated  from  the  water  with  which  it  has  been 
emulsified.  It  is  then  placed  in  the  cold  to  set.  The  water  may  be 
now  either  centrifugalised  or  placed  in  sedimentation  flasks,  and  the 
deposit  examined  for  bacteria. 

The  Uses  of  Bacteria  in  Dairy  Produce 

In  considering  the  relation  of  bacteria  to  milk,  we  found  that 
many  of  the  species  present  were  injurious  rather  than  otherwise, 
and  when  we  come  to  consider  bacteria  in  dairy  products,  like  butter 
and  cheese,  we  find  that  the  dairyman  possesses  in  them  very  powerful 
allies.  "Within  recent  years  almost  a  new  industry  has  arisen  owing 
to  the  scientific  application  of  bacteriology  to  butter  and  cheese 
making. 

1.  Bacteria  in  Butter-Making 

As  a  preliminary  to  butter-making,  the  general  custom  in  most 
countries  is  to  subject  the  cream  to  a  process  of  "  ripening."  As  we 
have  seen,  cream  in  ordinary  dairies  and  creameries  invariably 
contains  some  bacteria,  a  large  number  of  which  are  in  no  sense 
injurious.  Indeed,  it  is  to  these  bacteria  that  the  ripening  and 
flavouring  processes  are  due.  They  are  perfectly  consistent  with 
the  production  of  the  best  quality  of  butter.  The  aroma  of  butter, 
as  we  know,  controls  in  a  large  measure  its  price  in  the  market. 
This  aroma  is  due  to  the  decomposing  effect  upon  the  constituents 
of  the  butter  of  the  bacteria  contained  in  the  cream.  In  the  months 
of  May  and  June  the  variety  and  number  of  these  types  of  bacteria 
are  decidedly  greater  than  in  the  winter  months,  and  this  explains 
in  part  the  better  quality  of  the  butter  at  these  seasons.  As  a 
result  of  these  ripening  bacteria  the  milk  becomes  changed  and 
soured,  and  slightly  curdled.  Thus  it  is  rendered  more  fit  for  butter- 
making,  and  acquires  its  pleasant  taste  and  aroma.  It  is  then 
churned,  after  which  bacterial  action  is  reduced  to  a  minimum  or 
absent  altogether.  Sweet-cream  butter  lacks  the  flavour  of  ripened 


BUTTER-MAKING  243 

or  sour-cream  butter.  The  process  is  really  a  fermentation,  the 
ripening  bacteria  acting  on  each  and  all  of  the  constituents  of  the 
milk,  resulting  in  the  production  of  various  bye-products.  This 
fermentation  is  a  decomposition,  and  just  as  we  found  when  dis- 
cussing fermentation,  so  here  also  the  action  is  only  beneficial  if 
it  is  stopped  at  the  right  moment.  If,  for  example,  instead  of  being 
stopped  on  the  second  day,  it  is  allowed  to  continue  for  a  week,  the 
cream  will  degenerate  and  become  offensive,  and  the  pleasant  ripen- 
ing aroma  will  be  changed  to  the  contrary.  Speaking  generally, 
about  25  per  cent,  of  cream  bacteria  exert  a  favourable  effect  on 
butter,  and  10-15  per  cent,  a  deleterious  effect.  Many  of  the  former 
are  acid-producers,  and  are  widely  distributed. 

Bacteriologists  have  demonstrated  that  butters  possessing 
different  flavours  have  been  ripened  by  different  species  of  bacteria. 
Occasionally,  one  comes  across  a  dairy  which  seems  to  be  impregnated 
with  bacteria  that  improve  cream  and  flavour  well.  In  other  cases 
the  contrary  happens,  and  a  dairy  becomes  impregnated  with  a 
species  having  deleterious  effects  upon  its  butter.  Such  a  species 
may  be  favoured  by  unclean  utensils  and  dairying,  by  disease  of  the 
cow,  or  by  a  change  in  the  cow's  diet.  Thus  it  comes  about  that  the 
butter-maker  is  not  always  able  to  depend  upon  good  ripening  for 
his  cream.  At  other  times  he  gets  ripening  to  occur,  but  the  flavour 
is  an  unpleasant  one,  and  the  results  correspond.  It  may  be  bitter  or 
tainted,  and  just  as  certainly  as  these  flavours  develop  in  the  cream, 
so  is  it  certain  that  the  butter  will  suffer.  Fortunately,  the  bacterial 
content  of  the  cream  is  generally  either  favourable  or  indifferent  in 
its  action.  Thus  it  comes  about  that  the  custom  is  to  allow  the 
cream  simply  to  ripen,  so  to  speak,  of  its  own  accord,  in  a  vat 
exposed  to  the  influence  of  any  bacteria  which  may  happen  to  be 
around.  This  generally  proves  satisfactory,  but  it  has  the  great 
disadvantage  of  being  indefinite  and  uncertain.  Occasionally  it 
turns  out  wholly  unsatisfactory,  and  results  in  financial  loss.  Shortly, 
it  may  be  said  that  cream-ripening  assists  the  making  of  butter  in 
four  ways : — 

1.  Churning  is  easier  and  more  effectual. 

2.  The  yield  of  butter  is  increased. 

3.  The  butter  has  better  keeping  qualities. 

4.  The  flavour  and  aroma  are  more  satisfactory. 

Control  of  Ripening  Process. — There  are  various  means  at  our 
command  for  improving  the  ripening  process.  Perfect  cleanliness 
in  the  entire  manipulation  necessary  in  milking  and  dairying,  com- 
bined with  freedom  from  disease  in  the  milch  cows,  will  carry  .us  a 
long  way  on  the  road  towards  a  good  cream-ripening.  Eecently, 
however,  a  new  method  has  been  introduced,  largely  through  the 


244  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

work  and  influence  of  Professor  Storch  in  Denmark,  which  is  based 
upon  our  new  knowledge  respecting  bacterial  action  in  cream- 
ripening.  We  refer  to  the  artificial  processes  of  ripening  set  up  by 
the  addition  of  pure  cultures  of  favourable  germs.*  If  a  culture  of 
organisms  possessing  the  faculty  of  producing  in  cream  a  good  flavour 
be  added  to  the  sweet  cream,  it  is  clear  that  advantage  will  accrue. 
This  simple  plan  of  starting  any  special  or  desired  flavour  by  intro- 
ducing the  specific  micro-organism  of  that  flavour,  may  be  adopted 
in  two  or  three  different  ways.  If  cream  be  inoculated  with  a  large, 
pure  culture  of  some  particular  kind  of  bacteria,  this  species  will 
frequently  grow  so  well  and  so  rapidly  that  it  will  check  the  growth 
of  the  other  bacteria  which  were  present  in  the  cream  at  the  com- 
mencement and  before  the  "  starter "  was  added.  That  is,  perhaps, 
the  simplest  method  of  adding  an  artificial  culture.  But  secondly, 
it  will  be  apparent  to  those  who  have  followed  us  thus  far,  that  if 
the  cream  is  previously  pasteurised  at  70°  C.,  these  competing  bacteria 
will  have  been  mostly  or  entirely  destroyed,  and  the  pure  culture, 
or  "  starter,"  will  have  the  field  to  itself.  There  is  a  third  modifica- 
tion, which  is  sometimes  termed  ripening  by  natural  starters.  A 
"  natural  starter  "  is  a  certain  small  quantity  of  cream  taken  from  a 
favourable  ripening — from  a  clean  dairy  or  a  good  herd — and  placed 
aside  to  sour  for  two  days  until  it  is  heavily  impregnated  with  the 
specific  organism  which  was  present  in  the  whole  favourable  stock 
of  which  the  "  natural  starter "  is  but  a  part.  It  is  then  added  to 
the  new  cream,  the  favourable  ripening  of  which  is  desired.  Of  the 
species  which  produce  good  flavours  in  butter,  the  majority  are  found 
to  be  members  of  the  acid-producing  class ;  but  probably  the  flavour 
is  not  dependent  upon  the  acid.  The  aroma  of  good  ripening  is 
also  probably  independent  of  the  acid  production. 

Artificial  Ripening. — Of  all  the  methods  of  ripening — natural 
ripening,  the  addition  of  "natural  starters,"  the  addition  of  pure 
cultures  with  or  without  pasteurisation — there  can  be  no  doubt  that 
pure  culture  after  pasteurisation  is  the  most  accurate  and  reliable. 
The  use  of  "  natural  starters "  is  a  method  in  the  right  direction ; 
yet  it  is,  after  all,  a  mixed  culture,  and  therefore  not  uniform  in 
action.  In  order  to  obtain  the  best  results  with  the  addition  of 
pure  cultures,  Professor  Eussell  has  made  the  following  recom- 
mendations : — 

1.  The  dry  powder  of  the  pure  culture  must  be  added  to  a  small 
amount  of  milk  that  has  been  first  pasteurised,  in  order  to  develop 
an  active  growth  from  the  dried  material. 

2.  The  cream  to  be  ripened  must  first  be  pasteurised,  in  order  to 

*  Such  pure  cultures  for  such  purposes  are  in  the  United  States  termed 
"starters,"  because  they  start  the  process  of  special  ripening.  For  the  sake  of 
convenience  the  term  will  be  used  here. 


BUTTER-MAKING  245 

destroy  the  developing  organisms  already  in  it,  and  thus  be  prepared 
for  the  addition  of  the  pure  culture. 

3.  The  addition  of  the  developing  "starter"  to  the  pasteurised 
cream,  and  the  holding  of  the  cream  at  such  a  temperature  as  will 
readily  induce  the  best  development  of  flavour. 

4.  The  propagation  of  the  "starter"  from  day  to  day.     Afresh 
lot  of  pasteurised  milk  should  be  inoculated  daily  with  some  of  the  pure 
culture  of  the  previous  day,  not  with  the  ripening  cream  containing 
the  culture.    In  this  way  the  purity  of  the  "  starter  "  is  maintained  for 
a  considerable  length  of  time.    Those  "  starters  "  are  best  which  grow 
rapidly  at  a  comparatively  low  temperature  (60-75°  F.),  which  produce 
a  good  flavour,  and  which  increase  the  keeping   qualities   of   the 
butter.      Now,  whilst  it   is    true  that  the  practice  of  using  pure 
cultures  in  this  way  is  becoming  more  general,  very  few  species  have 
been  isolated  which  fulfil  all  the  desirable  qualities  above  mentioned. 
In  America,  "  starters "  are  preferred  which  yield  a  "  high "  flavour, 
whereas  in  Danish  butter  a  mild  aroma  is  more  common.    In  this  country, 
as  yet,  very  little  has  been  done,  and  that  on  an  experimental  scale 
rather  than  a  commercial  one.     In  1891  it  appears  that  only  4  per 
cent,  of  the  butter  exhibited  at  the  Danish  butter  exhibitions  was 
made  from  pasteurised  cream  plus  a  culture  "  starter  " ;  but  in  1895, 
86  per  cent,  of  the  butter  was  so  made.     Moreover,  such  butter 
obtained  the  prizes  awarded  for  first-class  butter  with   preferable 
flavour.     Different  cultures  will,  of  course,  yield  differently  flavoured 
butter.     If  we  desire,  say,  a  Danish  butter,  then  some  species  like 
"  Hansen's  Danish  starter  "  would  be  added ;  if  we  desire  an  American 
butter,  we  should  use  a  species  like  that  known  as  "  Conn's  Bacillus, 
No.  41."     But  whilst  these  are  two  common  types,  they  are  not  the 
only  suitable  and  effective  "  starters."      On  many  farms  in  England 
there  are   equally  good   cultures,  which,  placed   under   favourable 
temperatures  in  new  cream,  would  immediately  commence  active 
ripening.      A  good   lactic   acid   culture   for   dairy   use   (a)   should 
sour  cream  strongly  in  a  short  time,  (&)  should  be  able  to  thrive  at 
low  temperatures,  and  (c)  should  produce  a  favourable  taste  and  flavour 
in  the  butter  (Jensen). 

Professor  H.  W.  Conn,  who,  with  Professor  Eussell,  has  done  so 
much  in  America  for  the  advancement  of  dairy  bacteriology,  reports 
a  year's  experience  with  the  bacillus  to  which  reference  has  been 
made,  and  which  is  termed  No.  41.*  It  was  originally  obtained 
from  a  specimen  of  milk  from  Uruguay,  South  America,  which  was 
exhibited  at  the  World's  Fair  in  Chicago,  and  proved  the  most 
successful  flavouring  and  ripening  agent  among  a  number  of  cultures 
that  were  tried.  The  conclusions  arrived  at  after  a  considerable 
period  of  testing  and  experimentation  appear  to  be  on  the  whole 
*  Report  of  Storr's  Agricultural  Expt.  Sta.,  State  of  Connecticut,  1895. 


246  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

satisfactory.  A  frequent  method  of  testing  has  been  to  divide  a 
certain  quantity  of  cream  into  two  parts :  one  part  inoculated  with 
the  culture,  and  the  other  part  left  uninoculated.  Both  have  then 
been  ripened  under  similar  conditions,  and  churned  in  the  same  way ; 
the  differences  have  then  been  noted.  It  is  interesting  to  know 
that,  as  a  result  of  the  year's  experience,  creameries  have  been  able 
to  command  a  price  varying  from  half  a  cent  to  two  cents  a  pound 
more  for  the  "  culture  "  butters  than  for  the  uninoculated  butters.  The 
method  advised  in  using  this  pure  culture  is  to  pasteurise  (by  heating 
at  155°F.)  six  quarts  of  cream,  and  after  cooling,  to  dissolve  in  this 
cream  the  pellet  containing  bacillus  No.  41.  The  cream  is  then  set 
in  a  warm  place  (70°  F.),  and  the  bacillus  is  allowed  to  grow  for  two 
days,  and  is  then  inoculated  into  twenty-five  gallons  of  ordinary 
cream.  This  is  allowed  to  ripen  as  usual,  and  is  then  used  as  an 
infecting  culture,  or  "  starter,"  in  the  large  cream  vats  in  the  pro- 
portion of  one  gallon  of  infecting  culture  to  twenty-five  gallons  of 
cream,  and  the  whole  is  ripened  at  a  temperature  of  about  68°  F.  for 
one  day.  The  cream  ripened  by  this  organism  needs  to  be  churned 
at  a  little  lower  temperature  (say  52-54°  F.),  but  to  be  ripened  at 
a  little  higher  temperature  than  ordinary  cream  to  produce  the  best 
results.  Cream  ripened  with  No.  41  has  its  keeping  power  much 
increased,  and  the  body  or  grain  of  the  butter  is  not  affected.  More 
than  200  creameries  in  America  used  this  culture  during  1895,  which 
proves  that  its  use  for  the  production  of  flavour  in  butter  is  feasible 
in  ordinary  creameries,  and  in  the  hands  of  ordinary  butter-makers, 
provided  they  will  use  proper  methods  and  discretion.  More  recently, 
pasteurisation  has  fallen  into  abeyance,  and  the  use  of  artificial 
cultures  is  said  to  have  declined  in  America.  In  England,  with  few 
exceptions,  practically  nothing  has  been  done  in  a  commercial  way 
in  the  direction  of  artificial  "  starters." 


2.  Bacteria  in  Cheese-Making- 

The  cases  where  it  has  been  possible  to  trace  bacterial  disease  to 
the  consumption  of  butter  and  cheese  have  been  rare.  Notwith- 
standing this  fact,  it  must  not  be  supposed  that  therefore  cheese 
contains  few  or  no  bacteria.  On  the  contrary,  for  the  making 
of  cheese  bacteria  are  not  only  favourable,  but  actually  essential, 
for  in  its  manufacture  the  casein  of  the  milk  has  to  be  separated 
from  the  other  products  by  the  use  of  rennet,  and  is  then  col- 
lected in  large  masses  and  pressed,  forming  the  fresh  cheese.  In 
the  course  of  time  this  undergoes  ripening,  which  develops  the 
peculiar  flavours  characteristic  of  cheese,  and  upon  which  its  value 
depends. 

We  have  said  that  the  casein  is  separated  by  the  addition  of 


CHEESE-MAKING  247 

rennet,  which  has  the  power  of  coagulating  the  casein.  But  this 
precipitation  may  also  be  accomplished  by  allowing  acid  to  develop 
in  the  milk  until  the  casein  is  precipitated,  as  in  some  sour-milk  or 
cottage  cheeses.  The  former  method  is,  of  course,  the  usual  one  in 
practice.  It  has  been  suggested  that  the  bacteria  contained  in  the 
rennet  exert  a  considerable  influence  on  the  cheese,  but  this,  although 
rennet  contains  bacteria,  is  hardly  established.  It  is  not  here,  how- 
ever, that  bacteria  really  play  their  role.  After  this  physical  separa- 
tion, when  the  cheese  is  pressed  and  set  aside,  is  the  period  for  the 
commencement  of  the  ripening  process. 

That  bacteria  perform  the  major  part  of  this  ripening  process, 
and  are  essential  to  it,  is  proved  by  the  fact  that  when  they  are 
either  removed  or  opposed  the  curing  changes  immediately  cease.  If 
the  milk  be  first  sterilised  (Freudenreich),  or  if  antiseptics,  like 
thymol,  be  added  (Adametz),  the  results  are  negative.  It  is  not  yet 
known  whether  this  ripening  process  is  due  to  the  influence  of  a 
single  organism  or  not.  The  probability,  however,  is  that  it  is  to  be 
ascribed  to  the  action  of  that  group  of  bacteria  known  as  the  lactic 
acid  organisms.  Nor  is  it  yet  known  whether  the  peptonisation  of 
the  casein  and  the  production  of  the  flavour  are  the  results  of  one  or 
more  species.  Freudenreich  believes  them  to  be  due  to  two  different 
forms. 

However  that  may  be,  we  meet  with  at  least  four  common  groups 
of  bacteria  more  or  less  constantly  present  in  cheese-ripening,  either 
in  the  early  or  late  stages.  First,  there  are  the  lactic  acid  bacteria,  by 
far  the  largest  group,  and  the  one  common  feature  of  which  is  the 
production  by  fermentation  of  lactic  acid ;  secondly,  there  are  the 
casein-digesting  bacteria,  present  in  relatively  small  numbers ;  thirdly, 
the  gas-producing  bacteria,  which  give  to  cheese  its  honeycombed 
appearance ;  lastly,  an  indifferent  or  miscellaneous  group  of  extrane- 
ous bacteria,  which  were  in  the  milk  at  the  outset  of  cheese-making, 
or  are  intruders  from  the  air  or  rennet.  All  these  four  groups  may 
bring  about  a  variety  of  changes,  beneficial  and  otherwise,  in  the 
cheese-making. 

Russell  divides  the  ripening  process  into  three  divisions : — 

1.  Period  of  Initial  Bacterial  Decline  in  Cheese. — Where  the  green 
cheeses  were  examined  immediately  after  removing  from  the  press, 
it  was  usually  found  that  a  diminution  in  numbers  of  bacteria  had 
taken  place.      This  period  of   decline   lasts  but  a   short  time,  not 
beyond  the  second  day.     Lower  temperature  and  expulsion  of  the 
whey   would   account   f.or   this  general   decline   in   all    species    of 
bacteria. 

2.  Period  of  Bacterial  Increase. — Soon  after  the  cheese  is  removed 
from  the  press  a  most  noteworthy  change  takes  place  in  green  cheese. 
A  very  rapid  increase  of  bacteria  occurs,  confined  almost  exclusively 


248  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

to  the  lactic  acid  group.  This  commences  in  green  cheese  about  the 
eighth  day,  and  continues  more  or  less  for  twenty  days.  In  Cheddar 
cheese  it  commences  about  the  fifth  day,  reaches  its  maximum  about 
the  twentieth  day,  declines  rapidly  to  the  thirtieth  day,  and  gradu- 
ally for  a  hundred  following  days.  During  the  first  forty  days  of 
this  period  the  casein-digesting  and  gas-producing  organisms  are 
present,  and  at  first  increasing,  but  relatively  to  only  a  very  slight 
degree.  With  this  rapid  increase  in  organisms  the  curd  begins  to 
lose  its  elastic  texture,  and  before  the  maximum  number  of  bacteria 
is  reached  the  curing  is  far  advanced.  Freudenreich  has  shown  that 
acid  inhibits  the  growth  of  the  casein-digesting  microbes,  and  vice 
versa. 

3.  Period  of  Final  Bacterial  Decline. — The  cause  of  this  decline 
can  only  be  conjectured,  but  it  is  highly  probable  that  it  is  due  to  a 
general  principle  to  which  reference  has  frequently  been  made,  viz., 
that  after  a  certain  time  the  further  growth  of  any  species  of 
bacteria  is  prevented  by  its  own  products.  "We  may  observe  that 
the  gas-producing  bacteria  in  Cheddar  cheese  last  much  longer  than 
the  peptonising  organisms,  for  they  are  still  present  up  to  eighty 
days.  Professor  Russell  aptly  compares  the  bacterial  vegetation  of 
cheese  with  its  analogue  in  a  freshly-seeded  field.  "  At  first  multi- 
tudes of  weeds  appear  with  the  grass.  These  are  the  casein-digesting 
organisms,  while  the  grass  is  comparable  to  the  more  native  lactic 
acid  flora.  In  course  of  time,  however,  grass,  which  is  the  natural 
covering  of  soil,  '  drives  out '  the  weeds,  and  in  cheese  a  similar  con- 
dition occurs."  In  milk  the  lactic  acid  bacteria  and  peptonising 
organisms  grow  together;  in  ripening  cheese  the  former  eliminate 
the  latter. 

Artificial  Ripening. — We  have  seen  that  the  conclusion  generally 
held  respecting  these  lactic  acid  bacteria  is  that  they  are  the  main 
agents  in  curing  the  cheese.  Upon  this  basis  a  system  of  pure 
"  starters "  has  been  adopted,  the  characteristics  of  which  must  be 
as  follows : — (a)  The  organism  should  be  a  pure  lactic-acid-producing 
germ,  incapable  of  producing  gaseous  products ;  (&)  it  should  be  free 
from  any  undesirable  aroma ;  (c)  it  should  be  especially  adapted  for 
vigorous  development  in  milk.  The  "  starter  "  may  be  propagated  in 
pasteurised  or  sterilised  milk  from  a  pure  culture  from  the  labora- 
tory. The  advantages  accruing  from  the  uses  of  this  lactic  acid 
culture,  as  compared  with  cheese  made  without  a  culture,  are  that 
with  sweet  milk  it  saves  time  in  the  process  of  manufacture ;  that 
with  tainted  milk,  in  which  acid  develops  imperfectly,  it  is  an  aid  to 
the  development  of  a  proper  amount  of  acid  for  a  typical  Cheddar 
cheese ;  and  that  the  flavour  and  quality  of  such  cheese  is  preferable 
to  cheese  which  has  not  been  thus  produced.  Professor  Russell  is  of 
opinion  that  the  lactic  acid  organisms  are  to  be  credited  with  greater 


CHEESE-MAKING  249 

ripening  powers  than  the  casein-digesting  organisms,  but  it  must  not 
be  forgotten  that  these  two  great  families  of  bacteria  are  still  more 
or  less  on  trial,  and  it  is  not  yet  possible  finally  to  decide  on  either 
of  them.  Lloyd  holds  that  though  "  the  greater  the  number  of  lactic 
acid  bacilli  in  the  milk  the  greater  the  chance  of  a  good  curd,"  still 
"  this  organism  alone  will  not  produce  that  nutty  flavour  which  is  so 
much  sought  after  as  being  the  essential  characteristic  of  an  excellent 
Cheddar  cheese." 

There  are  three  difficulties  to  be  encountered  by  dairymen 
"starting"  a  ripening  by  the  addition  of  a  pure  culture.  First, 
there  is  the  initial  difficulty  of  not  being  able  to  use  pasteurised 
milk  for  cheese,  as  such  milk  is  uncoagulable  by  rennet  (Lloyd). 
Hence  it  is  impossible  to  avoid  some  contamination  of  the  milk 
previous  to  the  addition  of  the  culture.  Secondly,  the  continual 
uncontaminated  supply  of  pure  culture  is  by  no  means  an  easy 
matter.  Thirdly,  the  maintenance  of  a  low-temperature  cellar  to 
prevent  the  rapid  multiplication  of  extraneous  bacteria  will,  in 
some  localities,  be  a  serious  difficulty.  These  difficulties  have, 
however,  not  proved  insurmountable,  and  by  various  workers  in 
various  localities  and  countries  culture-ripening  of  cheese  is  being 


*  As  regards  the  Cheddar  cheese  industry  in  this  country,  Lloyd  arrives  at  the 
following  five  conclusions  as  a  result  of  investigation  :— 

1.  To  make  Cheddar  cheese  of  excellent  quality,  the  Bacillus  acidi  lactici  alone 
is   necessary  ;   other  germs   will  tend  to  make  the  work  more  rather  than  less 
difficult.     Hence  scrupulous  cleanliness  should  be  a  primary  consideration  of  the 
cheese-maker. 

2.  No  matter  what  system  of  manufacture  be  adopted,  two  things  are  necessary. 
One  is  that  the  whey  be  separated  from  the  curd,  so  that  when  the  curd  is  ground 
it  shall  contain  not  less  than  40  per  cent,  of  water,  and  not  more  than  43  per  cent.  ; 
the  other  point  is  that  the  whey  left  in  the  curd  shall  contain  developed  in  it  before 
the  curd  is  put  in  the  press  at  least  1  per  cent,  of  lactic  acid  if  the  cheese  is  required 
for  sale  within  four  months,  and  not  less  than  *8  per  cent,  of  lactic  acid  if  the  cheese 
is  to  be  kept  ripening  for  a  longer  period. 

3.  The  quality  of  the  cheeses  will  vary  with  the  quality  of  the  milk  from  which 
they  have  been  made,  and  proportionately  to  the  amount  of  fat  present  in  that 
milk. 

4.  "  Spongy  curd  "  is  produced  by  at  least  five  organisms,  and  one  of  these  is 
responsible  for  a  disagreeable  taint  found  in  curd.     They  occur  in  water.     Hence 
the  desirability  of  securing  clean  water  for  all  manipulative  purposes,  and  also  for 
the  drinking  purposes  of  the  milch  cow. 

5.  The  fact  that  certain  bacteria  are  found  in  certain  localities  and  dairies  is  due 
more  to  local  conditions  than  to  climatic  causes. 

It  is  needless  to  remark  that  these  conclusions  once  more  emphasise  the  fact 
that  strict  and  continual  cleanliness  is  the  one  desideratum  for  bacteriologically 
good  dairying.  That  being  secured  in  the  cow  at  the  milking,  in  the  transit,  and 
at  the  dairy,  it  is  a  comparatively  simple  step,  by  means  of  pasteurisation  and  the 
use  of  good  pure  cultures  of  flavouring  bacteria,  to  the  successful  application  of 
bacteriology  to  dairy  produce. 


250  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

Abnormal  Cheese-Ripening1 

Unfortunately,  from  one  cause  or  another,  faulty  fermentations 
and  changes  are  not  infrequently  set  up.  Many  of  these  may  be 
prevented,  being  due  to  lack  of  cleanliness  in  the  process  or  in  the 
milking ;  others  are  due  to  the  gas-producing  bacteria  being  present 
in  abnormally  large  numbers.  When  this  occurs  we  obtain  what  is 
known  as  "gassy"  or  inflated  cheese,  on  account  of  its  substance 
being  split  up  by  innumerable  cavities  and  holes  containing  carbonic 
acid  gas  or  sometimes  ammonia  or  free  nitrogen.  Some  twenty-five 
species  of  micro-organisms  have  been  shown  by  Adametz  to  cause 
this  abnormal  swelling.  In  severe  cases  of  this  gaseous  fermentation 
the  product  is  rendered  worthless,  and  even  when  less  marked  the 
flavour  and  value  are  much  impaired.  Winter  cheese  contains  more 
of  this  species  of  bacteria  than  summer.  Acid  and  salt  are  both 
used  to  inhibit  the  action  of  these  gas-producing  bacteria  and  yeasts, 
and  with  excellent  results.  The  character  of  the  gas  holes  in  cheese  is 
not  of  import  in  the  differentiation  of  species.  If  a  few  gas  bacteria 
are  present,  the  holes  will  be  large  and  less  frequent ;  if  many,  the 
holes  will  be  small,  but  numerous.  (Swiss  cheese  having  this 
characteristic  is  known  as  Nissler  cheese.)  Many  of  these  gas- 
producing  germs  belong  to  the  lactic  acid  group,  and  are  susceptible 
to  heat.  A  temperature  of  140°  F.  maintained  for  fifteen  minutes 
is  fatal  to  most  of  them,  largely  because  they  do  not  form 
spores. 

The  sources  of  the  extensive  list  of  bacteria  found  in  cheese  are 
of  course  varied,  more  varied  indeed  than  is  the  case  with  milk.  For 
there  are,  in  addition  to  the  organisms  contained  in  the  milk  brought 
to  the  cheese  factory,  the  following  prolific  sources,  viz.,  the  vats 
and  additional  apparatus ;  the  rennet  (which  itself  contains  a  great 
number) ;  the  water  that  is  used  in  the  manufacture. 

In  addition  to  the  abnormalities  due  to  gas,  there  are  also  other 
faulty  types.  The  following  chromogenic  conditions  occur:  red 
cheese,  due  to  a  micrococcus;  blue  cheese,  produced,  according  to 
Vries,  by  a  bacillus ;  and  black  cheese,  caused  by  a  copious  growth 
of  low  fungi.  Sitter  cheese  is  the  result  of  Tyrothrix  geniculatus 
(Duclatix),  or  the  Micrococcus  casei  amari  of  Freudenreich,  a  closely 
allied  form  of  Conn's  micrococcus  of  bitter  milk.  Sometimes  cheese 
undergoes  a  putrefactive  decomposition,  and  becomes  more  or  less 
putrid.  At  other  times  it  becomes  "  tainted."  These  latter  condi- 
tions, like  the  gassy  cheeses,  are  due  to  the  intrusion  of  bacteria 
from  without,  or  from  udder  disease  of  the  cow.*  Healthy  cows, 
clean  milking,  and  the  introduction  of  pure  cultures,  are  the  methods 

*  For  a  discussion  on  the  duration  of  life  of  the  tubercle  bacillus  in  cheese,  see 
Nineteenth  Ann.  Rep.  Bureau  of  Animal  Industry,  1902,  p.  217. 


POISONOUS  CHEESE  251 

to  be  adopted  for  avoiding  "diseases"  of  cheese  and  obtaining  a 
well-flavoured  article  which  will  keep. 

Finally,  there  is  poisonous  cheese  which  is  of  more  importance  to 
the  public  health  than  all  the  other  abnormal  conditions  of  cheese 
put  together.  In  1883  and  1884  there  occurred  in  Michigan,  U.S.A., 
an  outbreak  of  cheese-poisoning.  Three  hundred  persons  in  all  were 
affected,  and  the  illness  was  traced  by  Professor  V.  C.  Vaughan  to  a 
poisonous  ptomaine  present  in  the  cheese,  and  to  which  he  gave  the 
name  tyro-toxicon.  It  is  not  improbable  that  this  ptomaine  is  a 
product  of  bacterial  fermentation.  It  is  one  of  a  large  class  of 
substances  said  to  be  formed  by  the  action  of  bacteria  upon  nitro- 
genous compounds.  It  is  unstable,  and  easily  destroyed  by  the 
action  of  heat  and  moisture,  and  even  by  exposure  to  the  air.  Being 
present  in  small  quantities  only,  it  has  never  been  isolated  in  suffici- 
ently large  quantities  to  allow  of  its  composition  being  definitely 
determined.  Tyro-toxicon  has  been  proved  to  be  a  violent  poison 
both  to  man  and  the  lower  animals.  A  minute  portion  consumed 
by  a  child  produced  sickness  and  diarrhoea  in  a  manner  almost 
identical  with  cholera  infantum  (Vaughan).  Similar  symptoms  were 
obtained  with  cats  and  dogs.  Vaughan  found  that  three  months  are 
required  for  the  formation  of  tyro-toxicon  in  milk  kept  in  tightly- 
stoppered  bottles;  but  under  certain  circumstances,  and  in  the 
presence  of  butyric  fermentation  in  milk,  the  poison  is  produced  in 
about  eight  or  ten  days.  Similar,  and  possibly  identical,  poisons 
occasionally  occur  in  cream,  rancid  butter  and  milk  (lacto-toxieon  and 
diazo-benzol).  They  have  the  same  poisonous  effects.  Vaughan  has 
isolated  a  microbe  growing  readily  on  ordinary  culture  media  and 
upon  fruit  and  vegetables.  This  micro-organism,  it  is  considered, 
may  be  the  agent  producing  tyro-toxicon,  but  the  bacteriology  of  the 
subject  has  not  been  worked  out. 

The  writer  investigated  a  similar  outbreak  due  to  tyro-toxicon  in 
Dutch  cheese  in  London  in  1901.*  Seventeen  persons  were  affected. 
The  symptoms  of  illness  in  all  these  17  cases  occurred  in  from  two 
to  eight  hours  after  eating  the  cheese  in  question,  which  came  from 
the  same  consignment.  Moreover,  the  symptoms  were  similar, 
namely,  epigastric  pain,  rigors,  vomiting,  diarrhoea,  prostration,  and 
some  fever.  The  degree  of  sickness  does  not  appear  to  have 
depended  upon  the  amount  of  cheese  eaten.  There  was  no  death 
attributed  to  the  poisoning,  and  in  general  the  symptoms  appear  to 
have  passed  off  in  the  course  of  forty-eight  hours.  A  short  incuba- 
tion period  suggests  that  the  poison  was  "  available "  in  the  cheese, 
as  a  product  of  previous  changes,  possibly  bacterial,  set  up  therein. 
A  long  incubation  period  between  eating  the  food  and  symptoms  of 
poisoning  would  suggest  that  the  persons  affected  had  consumed,  not 
*  Report  on  the  Pullic  Health  of  Fimbury,  1901,  pp.  110-116. 


252  BACTERIA  IN  MILK  AND  MILK  PRODUCTS 

the  products  of  bacteria,  but  the  bacteria  themselves,  which  had  then 
taken  some  little  time  to  produce  their  injurious  effects  in  the 
persons  eating  the  food.  In  the  present  case  the  incubation  period 
was  comparatively  short,  and  the  acuteness  of  the  symptoms  did  not 
appear  to  have  a  direct  relationship  to  the  amount  of  cheese  eaten.* 

*  For  a  general  discussion  and  bibliography  of  this  subject,  see  Die  Milch 
und  ihre  Bedeutung  fur  Volkswirtschaft  und  Volksgesundheit,  Hamburg,  1903, 
pp.  345-357. 


CHAPTEE  VIII 

BACTERIA   IN   OTHER   FOODS 

1.  Shell-fish  :  Oysters,  Cockles,  Clams,  and  their  Relation  to  Disease ;  Symptoms  of 
Oyster-borne  Disease ;  Channels  of  Infection  ;  Preventive  Methods — 2.  Meat 
Poisoning ;  Tuberculous  Meat — 3.  Ice-cream  and  Ice — 4.  Bacterial  Infection 
of  Bread— 5.  Miscellaneous  Foods,  Watercress,  etc. 

IN  this  chapter  the  occurrence  and  significance  of  bacteria  in  shell- 
fish, meat,  ice-cream,  and  bread  will  be  considered. 

1.  Shell-fish 

Shell-fish  have  recently  claimed  the  attention  of  bacteriologists, 
owing  to  the  outbreak  of  typhoid  and  other  epidemics  apparently 
traceable  to  oysters. 

Oysters. — It  was  not  till  1880  (Cameron)  that  any  substantial 
evidence  was  forthcoming  to  establish  the  view,  which  had  previously 
been  promulgated  (by  Pasquier  in  1816),  that  oysters  and  other 
shell-fish  might  convey  the  infection  of  typhoid  fever.  In  1893 
oysters  came  under  the  suspicion  of  Sir  Eichard  Thorne  Thome 
as  concerned  in  the  diffusion  of  scattered  cases  of  cholera  in  Eng- 
land, and  he  reported  on  the  risks  of  consuming  shell-fish  culti- 
vated at  sewage  outfalls.*  In  the  spring  of  1894  Dr  Newsholme 
reported  to  the  Corporation  of  Brighton  the  particulars  of  a  number 
of  cases  of  typhoid  fever  which  were  apparently  attributable  to  the 
consumption  of  oysters  obtained  from  layings  grossly  contaminated 
by  sewage.  At  the  end  of  the  same  year  an  outbreak  of  typhoid 
fever  occurred  at  the  Wesleyan  University,  in  the  State  of  Con- 
necticut, U.S.A.,  and  an  investigation  was  made  by  Professor  H. 

*  "  On  Cholera  in  England  in  1893,"  Local  Government  Board  Report,  1894. 


254  BACTERIA  IN  OTHER  FOODS 

W.  Conn,  who  found  that  the  only  channel  of  infection  was  the 
consumption  of  oysters  served  at  certain  college  suppers.  These 
oysters  had  been  obtained  from  dealers  at  Middletown,  and  had  been 
cultivated  on  oyster-beds  in  the  region  of  a  sewage  outfall.  The 
facts  briefly  were  these :  Two  cases  of  typhoid  fever  occurred  in  a 
house  discharging  into  a  certain  sewer ;  the  outfall  of  the  sewer  was 
in  immediate  proximity  to  an  oyster-bed,  from  which  oysters  were 
taken  for  consumption  at  the  college  suppers ;  23  cases  of  typhoid 
fever  followed  among  the  students  who  attended  the  suppers  at 
which  the  oysters  were  eaten,  but  these  cases  were  limited  to  three 
out  of  seven  fraternities;  the  only  article  of  food  used  by  the 
three  implicated  fraternities,  and  not  by  the  other  four,  was  raw 
oysters  from  the  polluted  consignment;  and  lastly,  some  of 
the  same  consignment  were  consumed  at  Amherst  College,  and  an 
outbreak  of  typhoid  fever  occurred  among  those  who  consumed 
them.* 

This  outbreak  furnished  evidence  almost  equal  to  a  series  of 
experiments  designed  with  the  object  of  proving  the  possibility  of 
the  transmission  of  typhoid  fever  by  oysters.  It  served  also  to 
stimulate  inquiry,  and  since  its  occurrence  a  number  of  outbreaks 
have  been  traced  to  a  similar  source.  Sir  William  Broadbent 
described  several  such  cases  in  1895,  and  Dr  Newsholme  continued 
to  follow  the  matter  up,  and  reported  that  in  1894  38'2  per  cent., 
in  1895,  33-9  per  cent.,  in  1896,  31'8  per  cent.,  and  in  1897,  307  per 
cent.,  of  the  total  cases  of  typhoid  fever  originating  in  Brighton 
were  caused  by  sewage-contaminated  shell-fish.  Between  midsummer 

1893  and  the  end  of  1902,  630  cases  of  enteric  fever  occurred  at 
Brighton,  of  which  Dr  Newsholme  states  226  or  36  per  cent,  were 
caused   by   sewage-polluted   shell-fish,    152   cases   being    traced    to 
oysters  and  the  remainder  to  other  kinds  of  shell-fish.-j-    In  1896  Dr 
Bruce  Law  reported  an  outbreak  of  typhoid  fever  at  Southend,  in 
which  certain  cases  had  apparently  been  due  to  the  same  vehicle 
of   infection.      In    the    same    year    Chantemesse   described   to   the 
Academy  of  Medicine   an  outbreak   at  Saint-Andre,  in   the  Medi- 
terranean Department  of  Herault,  which  was  caused  by  a  barrel 
of    oysters    derived    from    contaminated    oyster  -  beds    at    Cette. 
Fourteen  persons   eating  these  oysters   in  an   uncooked  condition 
contracted  typhoid  fever,  or  a  disease  simulating  it.     Evidence  of 
the  same  character  as  that  recorded  in  the  above  cases  was  forth- 
coming from  Brightlingsea  (Buchanan),  Chichester  (Theodore  Thom- 
son), Belfast  (Jaffe),  Southend-on-Sea  (Foulerton,  Nash),  Yarmouth, 

*  Seventeenth  Annual  Report  of  the  State  Board  of  Health  of  Connecticut,  U.S.A., 

1894  ;  New  York  Medical  Record,  1894  ;  Report  of  Medical  Officer  to  Local  Govern- 
ment Board,  1894-95. 

t  Report  on  Health  of  Brighton,  1902,  p.  45. 


OYSTERS  AND  TYPHOID  255 

Exeter,  Blackpool,  and  other  places.  In  1902  occurred  the  outbreaks 
of  typhoid  fever  and  similar  illnesses  at  Winchester  and  Southampton, 
following  upon  the  consumption  (at  mayoral  banquets)  of  oysters 
derived  from  some  oyster-beds  at  Emsworth.  From  the  same  beds  at 
the  same  time  oysters  were  obtained  which  apparently  caused  cases  of 
the  disease  at  Portsmouth,  Brighton,  Yentnor,  Hove,  and  Eastbourne. 
The  matter  was  inquired  into  by  Dr  Tiinbrell  Bulstrode,  who  found 
that  at  Winchester,  out  of  a  total  of  134  guests  at  the  banquet,  62 
or  46 '3  per  cent,  were  attacked  with  illness;  and  at  Southampton 
mayoral  banquet,  out  of  132  guests,  55  or  41*6  per  cent,  were  attacked 
with  illness.  Eleven  of  these  cases  were  enteric  fever,  and  44  were 
cases  of  gastro-enteritis.  In  the  two  outbreaks  266  persons  were 
guests,  21  (or  7*8  per  cent.)  were  attacked  with  enteric  fever,  and  118 
(or  44'3  per  cent.)  suffered  from  other  illness.  All  those  who  had  no 
oysters  escaped  enteric  fever.  After  a  minute  inquiry  Dr  Bulstrode 
came  to  the  following  conclusion : — (a)  Two  mayoral  banquets  occur  on 
the  same  day  in  separate  towns  several  miles  apart ;  (&)  in  connection 
with  each  banquet  there  occurs  illness  of  analogous  nature  attacking, 
approximately  speaking,  the  same  percentage  of  guests  and  at  cor- 
responding intervals ;  (c)  at  both  banquets  not  every  guest  partook 
of  oysters,  but  all  those  who  suffered  enteric  fever,  and  approxi- 
mately all  those  who  suffered  other  illness,  did  partake  of  oysters, 
the  exceptions  to  this  rule  appearing  insignificant  when  all  the  facts 
are  marshalled;  (d)  oysters  derived  directly  from  the  same  source 
constituted  the  only  article  of  food  which  was  common  to  the  guests 
attacked;  and  (e)  oysters  from  this  source  were  at  the  same  time 
and  in  other  places  proving  themselves  competent  causes  of  enteric 
fever.  It  may  be  added  that  the  oyster-beds  at  Emsworth  from 
which  the  implicated  oysters  were  obtained  are  in  immediate 
proximity  to  the  outfall  of  the  Emsworth  sewers,  and  had  for 
several  years  been  known  to  be  contaminated  beds.* 

In  1902  there  also  occurred  an  outbreak  of  typhoid  fever  at 
Mistley  and  Bardfield  in  Essex,  which  was  shown  to  be  due  to  oysters, 
in  which  only  a  small  portion  of  the  oysters  appears  to  have  been 
capable  of  causing  illness,  and  the  nature  of  the  illness  varied  from 
a  mere  feeling  of  nausea  and  weakness  to  a  fatal  attack  of  typhoid.f 

In  1902  and  1903  further  evidence  was  forthcoming  from  various 
sources  which  went  to  show  the  intimate  and  apparently  causal 
relationship  between  the  consumption  of  polluted  shell-fish  and 
typhoid  fever.  Dr  Nash,  of  Southend-on-Sea,  states  that  in  1902 
only  0*4  per  cent,  of  the  cases  (501)  of  notifiable  infectious  diseases 

*  Special  Report  to  the  Local  Government  Board,  14th  May  1903,  by  Dr  H. 
Timbrell  Bulstrode.  See  also  Foulerton's  Report  on  the  Pollution  of  Tidal  Fishing 
Waters  by  Sewage,  1903,  pp.  31-37. 

t  Report  of  Medical  Officer  of  Essex  County  Council,  1902,  pp.  53-60. 


256  BACTERIA  IN  OTHER  FOODS 

other  than  typhoid  fever  occurred  in  persons  who  had  eaten  shell-fish, 
whereas  54  per  cent,  of  the  cases  of  typhoid  fever  occurred  in  persons 
who  had  recently  eaten  shell-fish.  In  1903  the  comparative  figures 
were  2  per  cent,  and  90  per  cent,  respectively.* 

Evidence  necessary  to  prove  Contamination  of  Oysters. — Evidence 
of  contamination  of  oyster-layings  by  sewage  must  be  sought  in 
three  directions : — 

(1)  There  must  be  personal  inspection  of  the  neighbourhood  and 
surroundings  of   the   layings   and   storage   ponds.     The  immediate 
sanitary  circumstances  may  be  such  that  a  definite  conclusion  can 
be  come  to  that  the  locality  is  dangerously  unfit  for  the  purposes  of 
the  oyster  industry,  and  no   further   examination   by  chemical   or 
bacteriological  methods   will   be  necessary.      On  the   other   hand, 
local  inspection  may  not  reveal  any  probable  source  of  dangerous 
sewage  contamination ;  and  to  test  the  matter,  it  may  be  necessary 
to  make  further  examination  in  order  to  detect  traces  of  pollution 
which  may  have  arisen  from  sources  of  contamination  which  were 
not  obvious  to  the  eye. 

(2)  In  the  event  of  the  results  of  inspection  being  satisfactory, 
the  next  step  is  to  examine  the  water  in  which  the  oysters  are  laid, 
in  order  to  ascertain  whether  the  chemical  or  bacteriological  evidence 
of  sewage  contamination  is  sufficiently  strong  to  enable  one  to  say, 
in  spite  of  the  local  inspection,  that  the  sewage  is  not  sufficiently 
diluted  and  purified  to  obviate  all  possible  danger. 

(3)  A  bacteriological   examination   of   the  molluscs  themselves 
must   be  made,  in  order  to  ascertain  whether   they  contain   those 
bacteria   which   are   ordinarily   associated   with    contamination    by 
sewage. 

It  is  hardly  needful  to  add  that,  in  order  to  establish  the  fact  that 
infection  has  occurred  or  may  occur  from  the  consumption  of  polluted 
oysters,  it  is  necessary  to  prove  disease  in  persons  or  animals  who 
have  eaten  some  of  the  oysters.  In  any  inquiry  of  this  kind  it  is 
essential  to  take  into  consideration,  (a)  clinical  evidence,  (b)  the 
history  and  circumstances  of  each  case,  and  (c)  the  exclusion  of 
all  other  possible  causes. 

Symptoms  of  Oyster-Poisoning.  —Obviously,  the  diseased  conditions  set  up  by 
the  consumption  of  polluted  oysters  will  vary  according  to  circumstances.  If  the 
pollution  be  the  specific  infection  of  typhoid  fever,  the  clinical  disease  of  typhoid 
fever  will  supervene.  The.  same  applies  to  cholera.  But  in  many  cases  on  record 
the  illness  resulting  has  been  of  a  less  specific  character,  and  has  simulated  gastro- 
enteritis, colic,  certain  nervous  conditions,  and  so  on.  Hence  it  may  be  desirable 
to  make  a  provisional  classification  as  follows : — 

(a)  Nervous  Conditions,  which  Mosny  likens  to  curare  poisoning.  This  type  is 
rare,  always  severe,  and  generally  fatal. 

(ft)  Gastro-Enteritis. — This  group,  which  is  one  of  the  most  common  and  least 

*  Public  Health,  1903  (November),  pp.  81  and  82. 


OYSTERS  AND  TYPHOID  257 

fatal,  includes  colic,  nausea,  vomiting,  with  more  or  less  prostration.     The  onset  is 
generally  sudden,  and  the  attack  lasts  a  comparatively  short  time. 

(c)  Dysenteric  Symptoms  may  occur  which  simulate  the  symptoms  of  group  (6), 
but  are  more  severe.    In  this  group,  between  it  and  (6),  may  be  classified  the  cholera- 
like  conditions  which  sometimes  occur. 

(d)  Specific  Disease,  such  as  typhoid  fever  or  cholera.* 

In  all  cases  there  is,  of  course,  an  incubation  period,  which  is  usually  longer  in 
duration  than  that  occurring  in  ptomaine  poisoning. 

Infection  of  Oysters. — The  mode  of  infection  of  oysters  by 

pathogenic  bacteria  is  briefly  as  follows: — The  sewage   of   certain 

coast  towns  is  passed  untreated  into  the  sea.     At  or  near  the  outfall, 

oyster-beds  are  laid  down  for  the  purpose  of  "fattening"  oysters. 

Thus  they  become  contaminated  with  saprophytic  and  pathogenic 

germs  conte^"     u  the  sewage.     It  will  be  at  once  apparent  that 

sevpi"*1    . '  *  UT     .dry  questions  require  attention  before  any  deductions 

-«vvix  as  to  whether  or  not  oysters  convey  virulent  disease 

^sinners. 

The  precise  conditions  which  render  one  locality  more  favourable 
than  another  in  respect  to  oyster  culture  are  not  fully  known.  But 
it  has  been  observed  that  they  do  not  flourish  in  water  containing 
less  than  3  per  cent,  of  salt.  Hence  they  are  absent  from  the 
Baltic  Sea,  which,  owing  to  the  fresh  river-water  flowing  into  it, 
contains  a  small  percentage  of  salt.  Oysters  appear,  in  addition,  to 
favour  a  locality  where  they  find  their  chosen  food  of  small  ani- 
malcula  and  particles  of  organic  matter.  Such  a  favourable  locality 
is  the  mouth  of  a  river,  where  tides  and  currents  also  assist  in 
bringing  food  to  the  oyster.  Unfortunately,  however,  in  a  crowded 
country  like  England,  such  localities  round  our  coast  are  frequently 
contaminated  by  sewage  from  outfalls.  Thus  the  oysters  and  the 
sewage  come  into  intimate  relation  with  each  other. 

Professor  Giaxa  carried  out  some  experiments  in  1889  at  Naples 
which  appeared  to  show  that  the  bacilli  of  cholera  and  typhoid 
rapidly  disappeared  in  ordinary  sea-water.  Other  observers  at  about 
the  same  time,  notably  Foster  and  Freitag,  arrived  at  an  opposite 
conclusion.  Klein  also  found  the  cholera  bacillus  four  days  after 
the  removal  of  the  oysters  from  water  purposely  contaminated  with 
them.  In  1894  Professor  Percy  Frankland,  in  a  report  to  the 
Koyal  Society,  declared  "that  common  salt,  whilst  enormously 
stimulating  the  multiplication  of  many  forms  of  water  bacteria, 
exerts  a  directly  and  highly  prejudicial  effect  on  the  typhoid  bacilli, 
causing  their  rapid  disappearance  from  the  water,  whether  water 
bacteria  are  present  or  not."  Boyce  and  Herdman  found  that  up 
to  a  certain  point  oysters  could  render  clear  sewage-contaminated 

*  The  general  question  of  mollusc  poisoning  is  treated  of  by  Mosny  in  the  Revue 
d' Hygiene,  December  1899  to  March  1900.  Mosny  also  furnished  the  .French 
Government  with  reports  on  the  subject 

R 


258  BACTERIA  IN  OTHER  FOODS 

water,  and  could  live  for  a  prolonged  period  in  water  rendered  opaque 
by  the  addition  of  faecal  matter.  They  also  proved  that  the  number 
of  organisms  in  the  pallial  cavity  and  rectum  of  oysters  which  had 
been  in  clear  water  was  much  less  than  occurred  in  oysters  laid 
down  in  proximity  to  a  sewer  outfall  (10  bacteria  in  the  former  case 
as  against  17,000  in  the  latter).  It  was  found  that  more  organisms 
were  present  in  the  pallial  cavity  than  in  the  rectum.  B.  typhosus 
could  be  identified  in  cultures  taken  from  the  water  of  the  pallial 
cavity  and  rectum  fourteen  days  after  inspection,  but  in  diminishing 
numbers.*  Several  important  links  in  the  chain  of  evidence 
remained  in  obscurity.  It  was  at  this  time,  when  the  matter  was 
admittedly  in  an  unsatisfactory  stage,  that  Dr  Cartwright  Wood 
made  his  experiments.-]-  "We  have  not  space  here  to  enter  into  this 
work.  But  his  conclusions  seem  to  have  been  amply  established, 
and  were  to  the  effect  that  typhoid  and  cholera  bacilli  could,  as  a 
matter  of  fact,  exist  over  very  lengthened  periods  in  ordinary  sea- 
water.  The  next  step  was  to  demonstrate  the  length  of  time  the 
bacilli  of  cholera  remained  alive  in  the  pallial  cavity  and  body  of 
the  oyster.  Dr  Wood  found  they  did  so  for  eighteen  days  after 
infection,  though  in  greatly  diminished  numbers.  This  diminution 
was  due  to  one  or  all  of  three  reasons :  (a)  the  effect  of  the  sea- 
water  already  referred  to  as  finally  prejudicial  to  bacilli  of  typhoid ; 
(b)  the  vital  action  of  the  body-cells  of  the  oyster;  (c)  the  wash- 
ing away  of  bacilli  by  the  water  circulating  through  the  pallial 
cavity. 

Broadly  it  may  be  said  that  the  same  principles  apply  to  the 
typhoid  bacillus.  It  can  live  in  sea- water,  probably,  for  three  to 
five  weeks  (Klein,  Boyce)  although  it  does  not  appear  to  multiply 
in  this  medium.  Oysters  infected  with  the  typhoid  bacillus  can 
retain  their  infective  properties  for  two  to  three  weeks,  and  even 
if  placed  in  running  sea-water,  may  not  lose  their  infective  properties 
for  some  days.  Mosny  suggested  any  period  from  one  to  eight 
days.  In  cockles  there  is  evidence  to  show  that  the  typhoid  bacillus 
thrives  and  even  multiplies,  and  these  shell-fish  are  not  rendered 
free  from  infection  by  being  laid  in  pure  water. 

It  will  have  been  noticed  that  up  to  the  present  we  have  learned 
that  typhoid  bacilli  can  and  do  live  in  sea-water,  and  also  inside 
oysters  up  to  eighteen  days,  but  in  ever-diminishing  quantities. 
The  question  now  arises :  What  is  the  influence  of  the  oyster  upon 
the  contained  bacilli?  Under  certain  conditions  of  temperature 
organisms  may  multiply  with  great  rapidity  inside  the  shell  of  the 
oyster.  Yet,  on  the  other  hand,  the  amoeboid  cells  of  the  oyster, 

*  Report  of  British  Association  for  Advancement  of  Science,  1895  ;  and  Thompson^ 
Yates  Laboratory  Report,  vol.  ii. 

t  Brit.  Med.  Jour.,  1896,  ii.,  p.  760  et  seq. 


OYSTERS  AND  TYPHOID  259 

the  acid  secretion  of  its  digestive  glands,  or  the  water  circulating 
through  its  pallia]  cavity,  may  act  inimically  on  the  germs.  Proof 
can  be  produced  in  favour  of  the  third  and  last-named  mode  by  which 
an  oyster  can  cleanse  itself  of  germs.  So  far,  then,  we  have  met  with 
no  facts  which  make  it  impossible  for  oysters  to  contain  for  a  lengthened 
period  the  specific  bacteria  of  disease.  Let  us  now  turn  to  their  oppor- 
tunity for  acquiring  such  disease  germs.  It  is  afforded  them  during 
the  process  of  what  is  termed  "  fattening."  By  this  process  the  body 
of  the  oyster  acquires  a  plumpness  and  weight  which  enhances  its 
commercial  value.  This  desired  condition  is  obtained  by  growing  the 
oyster  in  "  brackish  "  water,  for  thus  it  becomes  filled  out  and  medhani- 
cally  distended  with  water.  But  if  this  water  contain  germs  of  disease, 
what  better  opportunity  could  such  germs  have  for  multiplication  than 
within  the  body-cavity  of  an  oyster  ?  "  The  contamination  of  sea- 
water,  therefore,  in  the  neighbourhood  of  oyster-beds  may  undoubtedly 
lead  to  the  molluscs  becoming  infected  with  pathogenic  organisms  " 
(Wood).  Yet  we  have  seen  that,  apart  altogether  from  the  individual 
susceptibilities  or  otherwise  of  the  consumer,  there  are  in  the  series 
of  events  necessary  to  infection  many  occasions  when  circumstances 
would  practically  free  the  oysters  from  infection.  This  explains  the 
absence  of  uniformity  in  degree  of  contamination,  and,  coupled  with 
individual  susceptibility  and  degree  of  cooking,  the  absence  of 
uniformity  in  causing  outbreaks  of  disease. 

The  sources  of  pollution  of  oysters  are  not  the  fattening  beds 
alone.  The  native  beds  also  may  afford  opportunity  for  contamina- 
tion. Then,  in  packing  and  transit,  and  in  storage  in  shops  and 
warehouses,  there  is  frequently  abundant  facility  for  putrefactive 
bacteria  to  gain  entrance  to  the  shells  of  oysters. 

Dr  Klein's  researches  into  this  question  have  been  largely  con- 
firmatory of  the  facts  elicited  by  Dr  Cartwright  Wood.*  Despite 
the  tendency  of  the  bacilli  of  cholera  and  typhoid  to  die  out  quickly 
in  crude  sewage,  the  sewage  is  sufficiently  altered  or  diluted  at  the 
outfall  for  these  organisms  to  exist  there  in  a  virulent  state.  We 
may  give  Dr  Klein's  conclusions : — 

1.  That  the  cholera  and  typhoid  bacilli  are  difficult  of  demonstra- 
tion in  sewage  known  to  have  received  them. 

2.  That  both  organisms  may  persist  in  sea-water  tanks  for  two 
or   more  weeks,  the  typhoid   bacillus   retaining  its   characteristics 
unimpaired,  the  cholera  bacillus  tending  to  lose  them. 

3.  That  oysters  from  sources  free  from  sewage  pollution  con- 
tained no  bacteria  of  sewage  (e.g.  B.  coli  communis).     Subsequently 
to  these  experiments,  Klein   examined   172  oysters   from   various 
layings  to  which  no  sewage  gained  access,  and  B.  coli  was  absent 

*  Special  Report  of  the  Medical  Officer  to  the  Local  Government  Board  on  Oyster 
Culture,  etc.,  1896. 


260  BACTERIA  IN  OTHER  FOODS 

in  all  cases.*  The  Massachusetts  State  Board  of  Health  have 
recently  arrived  at  similar  results,  and  conclude  that  the  presence 
of  B.  coli  in  shell-fish  is  abnormal  and  due  to  contamination  either 
by  sewage  or  by  uncleanly  handling.  The  presence  of  B.  coli  is 
therefore  looked  upon  as  "  invariable  aid  "  in  determining  the  occur- 
rence of  pollution.^ 

4.  That  oysters  from  sources  exposed  to  risk  of  sewage  contami- 
nation did  contain  colon  bacilli  and  other  sewage  bacteria. 

5.  That  in  one  case  Eberth's  typhoid  bacillus  was  found  in  the 
body  and  liquor  of  the  oyster.     Nor  do  typhoid  bacilli  lose  activity 
or  virulence  by  passing  through  an  oyster. 

In  1902  Dr  Klein  had  occasion  to  examine  a  number  of  oysters 
in  connection  with  the  Winchester  outbreak  of  typhoid  fever,  to 
which  reference  has  already  been  made.  In  all  18  oysters  were 
examined  with  the  following  results : — (a)  Every  one  of  the  18  con- 
tained B.  coli,  (b)  3  out  of  the  18  contained  a  bacillus  belonging  to 
the  Gsertner-typhoid  group,  and  (c)  3  out  of  15  contained  the  spores 
of  B.  enteritidis  sporogenes.  All  these  oysters  came  from  the 
Emsworth  layings,  and  all  showed  contamination  with  excremen- 
titious  matter.  At  the  end  of  1902  and  beginning  of  1903,  Dr  Klein 
examined  25  different  sets  of  oysters,  only  7  sets  of  which  showed 
no  signs  of  pollution.^ 

Boyce  examined  140  samples  of  shell-fish  at  Liverpool  in  1902, 
and  found  B.  coli  present  in  104,  and  B.  enteritidis  sporogenes  in  10 
cases.  The  former  was  more  frequently  present  in  oysters  and 
mussels,  and  the  latter  in  cockles.  § 

In  1903  Mr  Foulerton  examined  a  number  of  oysters  derived 
from  suspicious  oyster-beds,  with  the  object  of  detecting  the  presence 
or  absence  of  bacteria  characteristic  of  sewage.  The  two  sewage 
organisms  which  he  selected  as  "  indication "  bacteria  were  B.  coli 
and  B.  enteritidis  sporogenes,  and  his  results  were  as  follows :  Out  of 
65  oysters  examined,  in  48  neither  bacillus  was  found;  in  5,  B. 
enteritidis  sporogenes  was  present  alone;  in  8,  B.  coli  was  present 
alone ;  and  in  4,  both  organisms  were  present.  Foulerton  attaches 
most  importance,  as  indication  of  recent  sewage  contamination,  to  the 
presence  of  B.  coli,  and  he  therefore  concludes  that  out  of  65  oysters 
12  or  19 '4  per  c.ent.  showed  evidence  of  recent  sewage  pollution. || 
In  a  second  series  of  27  oysters  B.  coli  was  found  in  4  instances,  or 
a  percentage  of  14*7. 

*  Brit.  Med.  Jour.,  1903,  i.,  p.  419. 

t  Thirty-fourth  Annual  Report  of  the  State  Board  of  Health  of  Massachusetts, 
1903,  pp.  260-264,  and  280. 

t  Report  of  Medical  Officer  of  Health,  City  of  London,  1902,  pp.  150-157. 

§  Report  on  Health  of  Liverpool,  1902,  p.' 173. 

II  The  Pollution  of  Tidal  Fishing  Waters  by  Sewage,  1903.  A  special  report  by 
A.  G.  R.  Foulerton,  F.R.C.S.,  D.P.H.,  pp.  42-49. 


OYSTERS  AND  TYPHOID  261 

Lastly,  the  results  of  the  investigations  carried  out  by  Dr 
Houston  for  the  Eoyal  Commission  on  Sewage,  tend  to  prove  that 
the  contamination  of  oysters  by  B.  coli  is  widespread  and  not  alto- 
gether dependent  on  sewage  contamination  of  the  oyster.  He 
examined  over  1000  oysters,  and  nearly  all,  from  whatever  laying 
they  were  taken,  contained  B.  coli  or  coliform  organisms.  This  did 
not  hold  good  as  regards  deep-sea  oysters  which  were  free  from  this 
organism  (and  spores  of  B.  enteritidis  sporogenes)  as  was  also  deep-sea 
water.  Houston  found  the  number  of  B.  coli  in  an  oyster  varied 
from  10  to  10,000  (in  10-15  c.c.),  and  the  contents  of  the  stomach 
of  the  oyster  contained  more  B.  coli  than  the  liquor  in  the  shell. 
Fewer  B.  coli  were  found,  as  a  rule,  in  oysters  stored  in  pure  waters, 
but  instances  occurred  where  the  number  of  such  bacilli  was  as 
great  as  in  oysters  from  contaminated  sources.* 

Such  is  the  bacteriological  evidence  down  to  recent  date,  and 
whilst  some  of  it  may  appear  to  be  of  a  conflicting  nature,  there  are 
certain  conclusions  which  may  be  drawn.  First,  the  presence  of  B. 
coli  in  oysters  must  be  judged  relatively.  Secondly,  topographical 
evidence  as  to  pollution  must  be  taken  in  conjunction  with  bacterial 
evidence.  Thirdly,  there  is  the  broad  general  fact  that  oysters 
ordinarily  grown  on  oyster-beds  contaminated  with  bacteria  may, 
and  do  on  occasion,  contain  the  virulent  specific  bacillus  of  typhoid, 
which  can  live  both  in  sea-water  and  within  the  shell  of  the  oyster. 
This  being  so,  the  risk  of  infection  of  typhoid  by  oysters  is  a  real 
one.  Yet  in  actual  occurrence  many  conditions  have  to  be  fulfilled. 
For,  in  addition  to  the  fact  that  the  oysters  must  be  consumed,  as  is 
usual,  uncooked,  the  following  conditions  must  also  be  present : — 

(a)  Each  infective  oyster  must  contain  infected  sewage,  which 
presupposes  that  typhoid  excreta  from  patients  suffering  from  the 
disease  have  passed  into  that  particular  crude  sewage,  and  have  not 
been  disinfected. 

(b)  The  infective  oyster  must  have  fed  upon  infected  sewage,  and 
still  contain  the  virus  in  its  substance. 

(c)  There  must  have  been  no  period  of  natural  cleansing  after 
"  fattening." 

(d)  The  oyster  must  then  be  eaten,  uncooked  or  undercooked, 
by  a  susceptible  person. 

Even  to  this  formidable  list  of  conditions  we  must  add  the 
further  remark  that,  owing  to  the  vitality  of  the  body-cells  of  the 
oyster  or  to  the  lessened  vitality  of  the  bacilli  of  cholera  and  typhoid, 
it  is  generally  the  case  that  the  tendency  of  these  organisms  is 
rather  to  decrease  and  die  out  than  live  and  multiply. 

*  Royal  Commission  on  Seioage  Disposal:  Fourth  Report  on  Pollution  of  Tidal 
Waters 'and  Contamination  of  Shell-fish,  1904,  vols.  i.  and  iii.  The  latter  volume 
contains  a  large  amount  of  information  as  to  bacteriological  technique,  etc. 


262  BACTERIA  IN  OTHER  FOODS 

We  shall  probably  maintain  a  satisfactory  balance  of  trutb  if  we 
place  alongside  these  facts  the  summary  of  the  Local  Government 
Board  Eeport.  "  There  can  be  no  doubt,"  wrote  Sir  Eichard  Thome, 
"  that  oysters  which  have  been  brought  into  sustained  relation  with 
the  typhoid  bacillus  are  liable  to  exhibit  that  microbe  within  the 
shell  contents,  and  to  retain  it  for  a  while  under  circumstances  not 
only  permitting  its  rapid  multiplication  when  transferred  again  to 
appropriate  media,  but  conserving  at  the  same  time  its  ability  to 
manifest  its  hurtful  properties."  *  The  Eoyal  Commission  on  Sewage 
Disposal  concludes,  that  at  the  present  time  it  is  undesirable  to  con- 
demn oysters  only  on  bacteriological  evidence  of  the  presence  of  B. 
coli.  At  present  topography  must  stand  before  bacteriology,  and  the 
condemnation  or  otherwise  of  oysters  must  be  judged  on  broad, 
common-sense  lines.  It  should  be  borne  in  mind  that  the  oyster 
trade  is  a  considerable  industry,  which  should  not  be  injured  except 
on  proved  and  substantial  evidence. 

Means  of  Prevention, — In  the  special  report  issued  by  the 
Local  Government  Board  in  1896,  on  oyster  culture,  which  had  been 
drawn  up  by  Dr  Timbrell  Bulstrode,  accounts  are  given  with 
diagrams  of  the  layings,  fattening  beds,  and  storage  ponds  used  in 
oyster  cultivation  in  various  counties  round  the  coast  of  England 
and  Wales.  Various  proposals  were  made  by  Dr  Bulstrode  for  the 
control  of  this  industry.  In  some  cases,  particularly  the  larger 
layings,  altering  the  position  of  the  fattening  beds  was  considered 
sufficient,  but  in  other  cases  nothing  short  of  a  complete  diversion  of 
the  sewers  and  drains,  or  withdrawal  of  existing  layings,  could  be 
regarded  as  sufticient.f  We  may  repeat  that  the  Eoyal  Commission 
on  Sewage  Disposal  urge  that  topographical  conditions  shall  be  taken 
along  with  bacteriological  evidence  in  arriving  at  a  decision  as  to  any 
oyster  layings,  and  under  the  present  circumstances  of  our  limited  know- 
ledge of  the  bacteriology  of  the  subject,  this  is  the  right  course  and 
should  assist  in  indicating  preventive  methods. 

From  what  has  been  said,  such  preventive  treatment  is  obvious  : — 
(1)  All  oyster  layings  and  shell-fish  beds  round  the  coast  should  be 
registered,  superintended,  and  inspected  by  the  sanitary  authority  or 
the  Government.  (2)  Local  Sanitary  Authorities  should  have  power 
of  control  over  oyster  layings  situated  in  their  district,  and  should 
be  enabled  to  prevent  the  sale  in  their  district  of  oysters  and  other 
molluscs  derived  from  sewage  -  contaminated  sources.  (3)  The 
importation  of  foreign  oysters,  grown  on  uncontrolled  beds,  should, 

*  Special  Report  to  Local  Government  Board  on  Oyster  Culture,  etc.,  1896.  This 
report  by  Dr  Timbrell  Bulstrode  is  probably  the  fullest  statement  yet  written  on  the 
question  as  it  affects  England. 

f  For  a  brief  record  of  the  attempts  at  legislation  on  this  subject,  see  Hrti. 
Jour.,  1903,  ii.,  p.  296  (Newsholme). 


COCKLES  263 

if  possible,  be  restricted  or  supervised.  (4)  Further,  as  a  protective 
measure  of  the  first  importance,  oysters  should  be  cleansed,  after 
fattening  on  a  contaminated  bed,  by  being  deposited  for  several 
weeks  at  some  point  along  the  coast  which  is  washed  by  pure  sea- 
water.  (5)  Retention  in  dirty- water  tanks,  in  uncleanly  shops  and 
warehouses,  should  also  be  prohibited. 

Other  shell-fish  than  oysters  do,  from  time  to  time,  cause 
epidemics  or  individual  cases  of  gastro-intestinal  irritation,  and  prob- 
ably contain  various  germs.  These  they  acquire  in  all  probability 
from  their  food,  which  by  their  own  choice  is  frequently  of  a  doubt- 
ful character. 

In  a  preliminary  inquiry  into  "Cockles  as  agents  of  Infectious 
Diseases,"  Dr  Klein  detected  the  B.  coli  in  3  out  of  8  cockles 
which  had  been  taken  from  a  foreshore  polluted  with  the  discharge 
from  a  sewer  outfall,  and  also  B.  enteritidis  sporogenes  in  4  of  them. 
No  typhoid  bacilli  were  detected.  In  8  raw  cockles  in  their 
shells  bought  from  a  street  hawker,  Dr  Klein  found  no  typhoid 
organisms,  but  B.  coli  was  found  in  5  out  of  the  8  cockles  and 
B.  enteritidis  sporogenes  in  4  out  of  the  8.*  In  subsequent  experi- 
ments Dr  Klein  came  to  the  conclusion  that  a  mussel  immersed  for 
twenty-two  hours  in  cholera-infected  water  retained  the  bacilli  of 
cholera  for  forty-eight  hours  after  immersion  in  clean  sea- water,  and 
the  same  may  be  said  in  respect  of  typhoid  infection.  Indeed,  evi- 
dence was  obtained  showing  that  the  typhoid  bacillus  could  multiply 
in  cockles.  He  also  showed  that  merely  pouring  boiling  water  over 
a  heap  of  shell-fish  did  not  necessarily  destroy  either  cholera  or 
typhoid  infection  contained  in  them.f  Since  the  time  of  these 
investigations,  a  number  of  outbreaks  of  disease,  including  enteric 
fever,  have  been  traced  to  the  consumption  of  mussels  and  cockles, 
and  it  has  been  shown  that  the  cooking  which  these  shell-fish 
undergo  is  not  sufficient  to  rid  them  of  poisonous  pollution. 

In  1902  several  cases  of  typhoid  occurred  in  Wandsworth  due 
to  infected  cockles,  and  a  number  of  similar  cockles  being  examined 
by  Dr  Klein  showed  the  presence  of  B.  coli  and  other  allied  forms, 
and  by  other  workers  (Lister  Institute)  during  the  same  year  the 
typhoid  bacillus  itself  is  stated  to  have  been  isolated  from  cockles 
derived  from  a  sewage-polluted  laying.^ 

In  1903  an  outbreak  of  typhoid  fever  occurred  in  Glasgow, 
which  was  traced  to  the  consumption  of  sewage-polluted  shell-fish 
at  a  neighbouring  seaside  town.  An  examination  of  a  number  of 
shell-fish  from  this  particular  locality  was  made  by  Dr  E.  M. 
Buchanan,  the  Corporation  bacteriologist,  who  reported  that — (1)  All 

*  Report  of  Medical  Officer  to  Local  Government  Board,  1899-1900,  p.  574. 

t  Ibid.,  1900-01,  pp.  564-71. 

£  Report  of  Medical  Officer  of  Health  of  City  of  London,  1902,  pp.  134-49. 


264  BACTERIA  IN  OTHER  FOODS 

the  edible  shell-fish  within  the  area  of  sewage  contamination  showed 
by  the  presence  of  virulent  B.  coli  excremental  pollution,  and  their 
consumption  must  therefore  be  regarded  as  highly  prejudicial  to 
health.  Cultures  of  the  species  of  B.  coli  killed  guinea-pigs  within 
eighteen  hours.  (2)  The  shell-fish  beyond  the  range  of  sewage 
contamination  were  found  to  be  normal  and  perfectly  safe  for  edible 
purposes.  (3)  Certain  shell-fish,  cockles  and  "  muscins "  (My a 
arenaria)  within  the  area  of  sewage  contamination  showed,  accord- 
ing to  Buchanan,  the  presence  of  the  bacillus  of  typhoid  fever  in 
great  number,  and  in  some  cases  almost  in  pure  culture,  and  the 
consumption  of  similarly  infected  shell -fish  by  holiday  visitors  in 
the  end  of  July  would  sufficiently  explain  the  outbreak  of  typhoid 
fever  which  occurred  amongst  them  after  return  to  their  own  homes 
in  Glasgow  and  elsewhere. 

As  regards  the  specificity  of  this  bacillus,  Buchanan  reports  that 
it  had  all  the  microscopical  and  cultural  characteristics  of  the 
typhoid  bacillus.  Further,  it  gave  the  characteristic  reaction  with 
human  typhoid  serum  and  with  serum  obtained  from  a  typhoid 
immunised  guinea-pig.  The  finding  of  this  bacillus  in  such  numbers, 
and  in  so  many  individual  shell-fish,  is  so  exceptional  that  it  was 
repeatedly  subjected  to  reliable  culture  tests  and  to  repeated  serum 
tests,  and  always  with  the  result  of  proving  its  general  identity  with 
the  typhoid  bacillus  obtained  from  a  case  of  typhoid  fever. 

A  third  apparent  instance  of  finding  the  typhoid  bacillus  may 
be  quoted.  In  1902-1903  Klein  examined  ten  samples  of  Leigh 
cockles,  and  found  every  one  of  them  showing  evidence  of  sewage 
pollution,  though  six  had  been  "  cooked."  One  of  the  uncooked  ones 
contained  B.  typhosus,  a  typical  typhoid  bacillus  agglutinating  with 
typhoid  blood  (Klein).  The  cooking  to  which  some  of  these  cockles 
were  submitted  must  have  been  perfunctory,  as  it  is  fairly  well  estab- 
lished that  60-61°  C.  kills  B.  coli.  Yet  this  organism  was  found  in 
two  instances  where  the  cockles  in  question  had  been  "boiled  con- 
tinuously for  one  minute,"  or  "  put  in  boiling  water  and  taken  out 
when  the  water  boiled  over,  time  in  water  three  and  a  quarter 
minutes."  * 

These  facts  reflect  a  new  and  not  reassuring  light  upon  the 
possibility  of  cockle  infection.  But  they  must  be  accepted  with 
great  reserve  until  very  fully  confirmed.  Dr  Bulstrode  in  his  report 
on  Oyster  Culture  in  1896  suggested  that  the  infection  by  cockles 
was  a  remote  contingency,  because  "  in  the  first  place  these  molluscs 
are,  as  a  rule,  only  eaten  after  being  cooked ;  and  in  the  second  place 
it  is  seldom  that  extensive  cockle  industries  are  carried  on  in  other 
localities  than  those  where  large  stretches  of  sand  are  exposed  at 
low  tide,  and  such  stretches  are  found  chiefly  on  the  actual  seashore 
*  Report  of  Medical  Officer  of  Health  of  City  of  London,  1902,  pp.  134-149, 


COCKLES  265 

or  quite  at  the  mouth  of  estuaries  far  away  from  sources  of  sewage 
contamination."  The  experience  at  Leigh-on-Sea,  Southend,  and 
other  places  seems  to  tell  a  different  tale,  and  it  is  evident  that 
the  shell-fish  may  be  grown  on  polluted  beds.  After  growth,  it  is 
true,  they  are  raked  into  hand-nets,  and  taken  to  the  cockle-sheds, 
and  here  are  plunged  into  coppers  of  boiling  water  in  the  nets,  after 
which  they  are  riddled  through  wide-meshed  sieves,  which  allow 
the  soft  parts  to  pass  through,  retaining  the  shells,  which  are 
deposited  in  heaps  for  sale  to  oyster  cultivators.  The  cockles  them- 
selves are  then  washed  in  about  five  changes  of  water,  to  the  last 
of  which  a  certain  quantity  of  salt  is  added.  Not  infrequently,  the 
same  water  is  used  in  all  the  washings.  The  so-called  boiling  is 
evidently  misleading.  Though  the  water  is  actually  at  the  boiling 
point,  the  cockles  are  plunged  in  in  a  mass,  and  for  a  short  time, 
and  it  by  no  means  follows  that  every  part  is  exposed  to  a  tempera- 
ture of  212°  F. 

Dr  Klein  has  shown  that  the  usual  method  of  cooking  only 
amounts  to  scalding,  and  cannot  be  relied  on  to  sterilise  micro- 
organisms. The  live  fish,  with  shells  tightly  closed,  are  held  in  a 
net  and  plunged  en  masse  into  a  vessel  containing  boiling  water. 
The  immersion  of  the  cold  mass  immediately  lowers  the  tempera- 
ture, and  when  in  the  course  of  two  or  three  minutes  it  begins  to 
boil  again,  the  net  is  lifted  out.  The  scalding  kills  the  fish  and 
causes  the  shells  to  open,  but  it  does  not  sterilise  the  contents. 
Dr  Klein  found  that  the  temperature  of  the  water  fell,  on  the  im- 
mersion of  the  fish,  from  100°  C.  to  65° ;  and  that  cooking  for  the 
usual  time  was  totally  inadequate  to  kill  the  micro-organisms.  Fish 
that  had  been  kept  in  typhoid  polluted  water  were  tested,  and  were 
found  to  be  swarming  with  live  bacilli  after  cooking.  Prolonged 
boiling  would,  no  doubt,  be  effective,  but  it  causes  the  fish  to 
shrivel  up  and  spoils  them  for  sale. 

Dr  Klein  then  suggested  that  cooking  by  steam  might  be  found 
an  efficient  steriliser  without  spoiling  the  fish  as  food.  It  is  well 
established  that  current  steam  is  much  more  penetrating  than 
boiling  water  for  purposes  of  disinfection,  and  it  is  always  used  in 
preference.  The  question  was  whether  an  exposure  sufficient  to 
sterilise  would  amount  to  over-cooking,  and  recent  experiments 
carried  out  in  the  kitchen  at  Fishmongers'  Hall  were  intended  to 
settle  that  point.  Cockles  and  mussels  were  cooked  in  a  steamer 
under  the  direction  of  Dr  Klein  in  the  presence  of  several  repre- 
sentatives of  the  trade,  who  examined  them  afterwards.  Two 
batches  were  cooked,  one  for  ten  minutes  and  the  other  for  five. 
The  steamer  used  was  a  fixed  vessel  some  2  feet  deep,  into  which 
steam  is  introduced  by  a  pipe  about  an  inch  from  the  bottom.  A 
layer  of  cockles  was  placed  at  the  bottom,  and  two  other  layers  on 


266  BACTERIA  IN  OTHER  FOODS 

trays  above  it.  In  the  top  tray  mussels  were  also  placed.  Some  of 
the  fish  were  spread  out  and  others  heaped  up.  The  results  were : — 
Ten  minutes — mussels  pronounced  spoilt,  and  useless  to  the  trade ; 
cockles  "  all  right "  in  upper  layers,  but  the  bottom  layer  overcooked. 
Five  minutes — mussels  "  all  right,"  and  cockles  better  than  the  ten 
minutes'  batch ;  the  upper  layers  "  could  not  be  better "  in  appear- 
ance and  flavour,  but  the  bottom  layer  was  again  pronounced 
somewhat  overcooked,  or  at  any  rate  less  satisfactory  than  the 
others.  No  doubt  the  steam  was  hotter  at  the  bottom  of  the  vessel 
and  the  exposure  greater.  The  bacterial  results  were  as  follows : — 
The  cockles  were  found  to  be  sterile  in  all  cases :  the  mussels  were 
also  found  to  be  sterile,  except  in  the  case  of  those  placed  in  heap  on 
the  top  layer  and  steamed  for  five  minutes.  Some  of  these  still 
retained  living  spores.  It  is  probable  that  if  exposed  to  the  more 
direct  action  of  the  steam  even  the  heaped  mussels  would  be 
completely  sterilised  by  five  minutes'  cooking,  without  impairing 
their  trade  value.  As  a  result  of  these  experiments  the  Fish- 
mongers' Company  was  reported  as  recommending  to  the  trade  the 
substitution  of  steaming  for  boiling. 

Many  other  similar  foods  have  been  implicated  in  the  spread  of 
disease.  Dr  Hamer  investigated  outbreaks  of  typhoid  fever  in 
London  in  1900  and  1903,  in  which  he  showed  the  extreme  prob- 
ability of  fried  fish  acting  as  the  Vehicle  of  infection.*  In  1900  Dr 
Plowright  traced  similar  infection  in  thirty  persons  to  polluted 
clams,  shell-fish  comparatively  little  known  in  this  country  as  an 
article  of  diet.f  Derived  from  sewage-polluted  layings,  clams  may 
readily  become  contaminated,  and  if  uncooked  may  convey  disease  to 
the  consumer. 

*  Ninth  Annual  Report  of  Medical  Officer  of  Health  of  Administrative  County  of 
London,  1900,  p.  37,  and  Appendix;  and  Special  Report,  No.  719,  issued  1904. 

f  The  clam  is  a  shell-fish  comparatively  little  known  in  this  country  as  an 
article  of  diet  except  to  the  dwellers  near  those  of  our  coasts  on  which  it  occurs. 
Belonging  to  the  Siphonidae  division  of  the  Conchiferse,  the  clam  (My a  arenaria'), 
like  its  ally  the  cockle,  is  found  abundantly  round  our  shores.  It  has,  however,  a 
wider  geographical  distribution,  being  found  in  the  Arctic  Regions,  where  it  con- 
stitutes an  important  article  of  food.  In  America  it  is  largely  consumed  in  Boston 
and  along  the  Massachusetts  seaboard.  The  clam  of  New  York  is  a  different  species 
(Venus  marcenaria).  It  has  a  remarkably  developed  syphon,  the  inhalant  and 
exhalent  tubes  being  joined  into  a  trunk-like  body  3  or  4  inches  in  length,  which  the 
animal  protrudes  in  an  upward  direction  towards  the  surface  of  the  mud.  The  clam 
itself  lies  buried  in  the  mud,  into  which  it  has  worked  itself  by  the  aid  of  its  muscular 
foot  to  a  depth  varying  from  8  to  18  inches.  The  currents  of  water  passing  in  and 
out  the  syphon  keep  open  the  vertical  burrow  the  creature  has  made,  while  the 
surface  of  the  mud  is  covered  by  the  tide,  but  where  this  recedes  and  the  mud 
becomes  dry,  the  position  of  the  clam  is  shown  by  a  small  round  depression  on  the 
surface.  In  Great  Britain  it  is  regarded  as  a  kind  of  inferior  oyster,  and  like  the 
last-named  is  preferred  uncooked  by  those  persons  who  are  really  fond  of  it  and  to 
whom  it  is  a  luxury.  More  generally,  clams  are  cooked  by  having  boiling  water 
poured  over  them,  and  being  allowed  to  remain  in  it  until  the  shells  open. 


ETIOLOGY  OF  MEAT-POISONING 


267 


2.  Meat 

Since  1880,  more  than  fifty  outbreaks  of  disease  have  been  traced 
to  the  consumption  of  unwholesome  or  diseased  meat.  In  1880 
occurred  the  well-known  "Welbeck  disease"  epidemic.  A  public 
luncheon  was  followed  by  severe  and,  in  some  cases,  fatal  illness. 
Seventy-two  persons  were  affected  and  four  died.  A  specific  bacillus 
was  isolated  by  Klein  from  the  cold  hams,  the  consumption  of 
which  caused  the  outbreak.  The  incubation  period  varied  from 
twelve  to  forty-eight  hours.  This  epidemic  drew  marked  attention 
to  the  whole  question  of  food-poisoning,  and  subsequent  epidemics 
were  very  thoroughly  investigated  by  the  aid  of  bacteriology.  Some 
of  the  better-known  outbreaks  may  be  tabulated  as  follows : — 


Date  of 
Occurrence. 

Place  of  Occurrence. 

No.  of 
Cases. 

Period  of 
Incubation  in 
Hours. 

Probable  Source  of 
Infection. 

1880 

Welbeck 

72 

12-48 

Cold  boiled  hams 

1881 

Nottingham 

15 

12-34 

Pork 

1882 

Oldham 

9 

4 

American  Tinned 

Pig's  Tongue 

1882 

Bishop  Stortford 

6 

24 

Beef 

1882 

Whitchurch 

20 

1-5 

Brawn 

1886 
1886 

Carlisle 
Ironbridge 

20 
12 

6-40 
6-12 

Ham  and  game  pie 
Veal  pies 

1887 

Retford 

80 

8-36 

Pork  brawn 

1888 

M  iddlesborough 

114 

... 

American  bacon 

1889 

Carlisle 

25 

24 

Pork  pies 

1891 

Portsmouth 

13 

14-17 

Cold  meat  pie 

1896 

Mansfield 

265 

18-24 

Potted  meat 

1898 

Oldham 

54 

48 

Veal  pies 

1899 

Nuneaton 

42 

12-48 

Pork  chitterlings 

1902 

Derby 

221 

4-24 

Pork  pie 

In  nearly  all  these  cases  the  general  symptoms  have  been  usually 
one  of  two  kinds,  namely,  conditions  simulating  gastro-enteritis,  or 
conditions  simulating  nervous  disease.  Each  of  the  outbreaks  have 
shown  more  or  less  clearly  the  characters  common  to  these  epi- 
demics : — 

1.  Simultaneous  attacks. 

2.  Similarity  of  symptoms  and  .post-mortem  signs. 

3.  A  history  of  infection  and  collateral  circumstances. 

The  common  symptoms  have  included  rigors,  faintness,  vomiting, 
diarrhoea,  abdominal  pain,  and  occasionally  skin  eruptions.  As  a 
rule,  certain  nervous  conditions  have  supervened,  such  as  giddiness, 
headache,  paralyses,  mental  depression,  etc.,  and  occasionally  these 
symptoms  have  been  predominant. 


268  BACTERIA  IN  OTHER  FOODS 

Meat-poisoning  appears  to  depend  not  upon  the  number  of 
bacteria  present  in  the  meat,  but  upon  the  particular  species  and 
their  products.  As  we  have  already  stated,  a  long  incubation  period 
generally  indicates  poisoning  by  bacteria,  and  a  short  incubation 
period  poisoning  by  products  (ptomaines,  toxins,  etc.).  In  1888, 
Gaertner  of  Jena  investigated  an  outbreak  of  disease  affecting  58 
persons  who  had  eaten  uncooked  meat.  One  of  the  unfortunate 
victims  died,  and  from  his  body,  as  well  as  from  the  meat,  Gaertner 
isolated  a  bacillus  which  he  called  the  B.  enteritidis,  an  organism 
allied  to  the  coli  group.  This  was  practically  the  starting-point  of 
accurate  bacteriological  investigation  into  this  group  of  epidemics 
(flcisclivergiftung,  Ger. ;  and  intoxications  aliment  air  cs,  FT.).  Since 
that  period,  the  B.  lotulinus  of  Ermengem,.and  certain  putrefactive 
bacteria,  have  been  held  responsible  for  causing  such  illnesses. 
More  than  twenty  different  species  of  bacteria  have  been  isolated 
from  tinned  meats  and  hams.  As  is  pointed  out  elsewhere  in  the 
present  volume,  there  is  evidence  that  the  infectious  properties  which 
food  acquires  frequently  in  summer,  and  which  give  rise  to  the 
ordinary  type  of  epidemic  diarrhoaa,  are  due  to  bacilli  belonging  to 
the  colon  group,  of  which  the  B.  coli  communis  of  Escherisch  and  the 
B.  enteritidis  of  Gaertner  are  the  two  extreme  types.  According  to 
Delepine,  the  varieties  of  those  bacilli  which  are  the  most  important 
sources  of  infection  are  those  which  resemble  the  bacillus  of  Gaertner, 
and  which,  therefore,  produce  no  permanent  acidity,  coagulation,  or 
distinct  smell  when  grown  in  milk.  Very  few  infectious  samples  of 
milk  give  a  distinct  acid  reaction,  so  that  absence  of  acidity  in  milk 
is  not,  as  generally  believed,  an  index  of  safety.  It  is  probable  that 
the  most  dangerous  kind  of  fsecal  infection  is  that  produced  by  matter 
containing  bacilli  resembling  Gaertner's  bacillus.  Such  an  infection 
is  probably  connected  with  the  existence  of  an  infectious  diarrhoeal 
disease  liable  to  occur  in  the  lower  animals  as  well  as  in  man. 

It  is  certain  that  bacilli  presenting  the  characters  of  the  ordinary 
B.  coli  communis  are  seldom  capable  of  producing  such  a  rapid 
infection  as  that  produced  by  the  B.  enteritidis,  or  by  closely-allied 
bacilli,  such  as  the  B.  enteritidis  Derliensis. 

The  last-named  organism  is  a  member  of  the  Gaertner  group 
isolated  by  Delepine  from  pork  pies,  the  consumption  of  which 
caused  the  Derby  illness  in  1902.  He  considered  the  presence  of 
this  bacillus  in  the  pork  pies  was  due  to  fecal  pollution  of  the 
meat  before  it  was  cooked,  and  that  the  central  parts  of  the  pies 
were  not  thoroughly  cooked.*  It  frequently  happens  in  these  cases 

*  Report  on  Outbreak  of  Food-Poisoning  in  Derby,  1902  (Howarlh  and  Delepine). 
In  this  reference,  and  in  Brit.  Med.  Jour.,  1898,  ii.'  pp.  1456-58  and  1797-1801,  and 
ibid.,  1899,  vol.  ii.,  pp.  791,  1367,  will  be  found  many  particulars  with  regard  to 
meat-poisoning,  its  symptoms,  prevention,  investigation,  etc. 


MEAT  269 

that  some  constituent  part  (such  as  jelly)  of  the  manufactured  article 
or  prepared  dish  is  really  the  polluted  portion. 

Bacteria  associated  with  Meat-Poisoning 

The  chief  organisms,  therefore,  which  have  been  considered  as  causally  related  to 
meat  (and  "  ptomaine  ")  poisoning  are  B.  coli  communis,  B.  enteritidis  sporogenes* 
B.  enteritidis  of  Gaertner,  and  B.  botulinus.  The  main  facts  respecting  these  organisms 
must  be  mentioned  here. 

(a)  B.  coli  communis  (see  p.  46). 

(b)  B.  enteritidis  sporogenes  (see  pp.  156  and  307). 

(c)  B.  enteritidis  of  Gaertner.      Isolated  by  Gaertner   in   1888   from    flesh   of 
diseased  cow  which  had  caused  illness  in  persons  eating  it.     Characters  similar  to 
B.  typhosus  (morphology,  motility,  and  staining  properties),  but  grows  more  rapidly 
in  gelatine;  fewer  flagella;   ferments   lactose  and  sometimes  dextrose;    does  not 
produce  indol  or  coagulate  milk ;  positive   neutral-red  reaction ;  in  litmus  whey  or 
litmus  broth,  acid  is  first  produced,  and  then  the  medium  becomes  distinctly  alkaline. 
Virulent  to  rodents  and  small  animals  (gastro-intestinal  symptoms,  haemorrhagic 
enteritis,  and  swelling  of  lymph  follicles).    B.  enteritidis  Derbiensis  of  Delepine  is  one 
of  the  members  of  the  Gaertner  group  of  enteritidis  bacilli.     B.  enteritidis  possesses 
no  spores,  and    therefore    cannot    stand    very   high    temperatures.       It  produces 
agglutinating  properties  in  the  blood  of  the  patient. 

(d)  B.  botulinus  (Ermengem).     This  bacillus  is  held  to  be  responsible  for  setting 
up  botulism.     Van  Ermengem  describes,  under  the  name  of  botulism,  a  state  brought 
about  by  the  ingestion  of  various  articles  of  food,  such  as  ham,  tinned  or  preserved 
foods,  oysters,   mussels,  etc.,  and  which  is   characterised  by  comparatively  slow 
onset  (twelve  to  twenty-four  hours  after  infection),  secretory  troubles,  paralysis  of 
certain   muscles,   particularly  tongue  and  pharynx,   dilatation  of   pupil,   aphonia, 
dysphagia,  constipation,  retention  of  urine,  absence  of  unconsciousness  and  of  fever, 
etc.     Van  Ermengem  has  found  that  these  symptoms  were  produced  by  a  bacillus, 
to  which  he  has  given  the  name  of  bacillus  botulinus.     Botulism  differs  considerably 
from  the  more  common  form  of  food-poisoning  with  which  we  are  acquainted  in 
England,  and  which  is  characterised  by  practically  the  same  symptoms  as  those  of 
epidemic  diarrhoea.     B.  botulinus  is  4-9  fj.  long  and   '9-12  /*  broad;  round,  slowly 
motile,  4-9  flagella.     Polar  spores  ;  killed  in  thirty  minutes  at  80°  C.      Liquefies 
gelatine ;  does  not  coagulate  milk ;  anaerobic ;  in  cultures  often  produces  gas  and 
a  sour,  rancid  odour.    Pathogenic  for  guinea-pigs,  rabbits,  and  the  other  small  animals 
(botulism). 

Preventive  methods.  —  Experience  of  meat-poisoning  outbreaks 
leads  to  the  conclusion  that  the  meat  has  contracted  its  poisonous 
properties  in  either  or  both  of  two  ways — (a)  putrefaction  or  unsound- 
ness  in  the  meat  itself;  (b)  unclean  manipulation  or  storage  in 
insanitary  conditions.  Generally  there  has  also  been  insufficient 
cooking.  The  methods  of  prevention  are  therefore  obvious.  Occasion- 
ally tinned  foods  cause  poisoning  owing  to  metallic  absorption,  and 
this  must  be  differentiated  from  bacterial  poisoning. 

There  is  another  class  of  meat  conditions  related  to  disease,  to 
which  reference  must  now  be  made,  viz.,  certain  conditions  occurring 
in  fresh  meat,  joints,  or  carcases.  It  is  well  known  that  the  meat 
substance  itself  does  not  frequently  contain  injurious  bacteria. 
They  may  nevertheless  occur  in  the  organs,  glands,  and  tissues 


270  BACTERIA  IN  OTHER  FOODS 

other  than  muscular,  and  when  present  set  up  during  life  the 
bacterial  diseases  of  animals,  or  after  death  putrefactive  changes. 
It  is  for  these  conditions  that  meat  is  "seized"  under  the  Public 
Health  Acts  as  unfit  for  food  of  man.  Such  conditions  may  be 
broadly  divided  into  two  kinds : — 

1.  Specific  Diseases  in  Meat,  such  as  tuberculosis,  anthrax,  swine 
fever,  actinomycosis,  milk  fever,  etc. 

2.  Decompositions    of    Meat    due    to    invasion    by  putrefactive 
organisms  after  death.     These  conditions   may   be   disposed   of   at 
once  by  saying  that  they  arise  commonly  as  a  result  of  keeping  meat 
too  long  under  conditions  likely  to  lead  to  putrefaction.     Unclean 
storage,   insufficient    preservation,   summer    weather,    and    similar 
circumstances  afford  the  opportunity  for  putrefactive  organisms  to 
perform  their  function.    The  signs  of  decomposing  meat  do  not  require 
explanation  or  elaboration.     They  are  mainly  three : — (a)  Smell  of 
putrefaction;   (&)  discoloration;  and  (c)  loss  of  elasticity  of  tissue 
which  becomes  doughy,  pits  on  pressure,  or  may  even  become  slimy 
or  soapy. 

The  chief  specific  diseases  which  occur  in  meat  are  dealt  with 
briefly  in  the  section  treating  of  the  relation  between  bacteria  and 
disease.  It  will,  therefore,  be  unnecessary  to  make  more  than  a 
passing  reference  in  this  place. 

Tuberculosis.1— This  disease  is  set  up  in  animals  by  the  tubercle 
bacillus,  which  is  either  identical  with  or  closely  allied  to  the  B. 
tuberculosis  of  Koch.  It  may  set  up  a  generalised  disease  affecting 
the  body  of  the  animal  more  or  less  completely,  or  it  may  set  up 
only  a  local  disease. 

The  Koyal  Commission  on  Tuberculosis,  in  the  report  which  they 
made  in  1898,  referred  to  the  degree  of  tubercular  disease  which 
should  cause  a  carcase,  or  part  thereof,  to  be  seized,  and  which  may 
be  accepted  broadly  as  indicative  of  general  and  local  tuberculosis. 
They  stated  as  follows : — 

"We  are  of  opinion  that  the  following  principles  should  be 
observed  in  the  inspection  of  tuberculous  carcases  of  cattle : — 


(a)  When  there   is   miliary  tuberculosis 

of  both  lungs  .         .      ....'-« 

(b)  When  tuberculous  lesions  are  present 

on  the  pleura  and  peritoneum 

(c)  When  tuberculous  lesions  are  present 

in  the  muscular  system,  or  in  the 
lymphatic  glands  embedded  in  or 
between  the  muscles  .  , 

(d)  When   tuberculous   lesions   exist   in 

any  part  of  an  emaciated  carcase     .  j 


Generalised  tuber- 
culosis is  present, 
and  the  entire 
carcase  and  all 
the  organs  may 
be  seized. 


TUBERCULOUS  MEAT  271 

/  \  ^Ttn       ji     i     •  -i     1  Localised   tubercu- 

(a)  When  the  lesions  are  confined  to  the        losig  ^  pregent) 


lungs  and  the  thoracic  lymphatic 
glands     .      "  . 

(b)  When  the  lesions  are  confined  to  the 
liver 


/  \  ™-u       J-T,     i     •                                   ^1      r  condemned, 

(c)  When  the  lesions  are  confined  to  the  -,    ,                      / 

pharyngeal  lymphatic  glands  .         .  .     .       Contain- 

(d)  When  the  lesions  are  confined  to  any  ing    tuberculous 


combination  of   the  foregoing,  but 
are  collectively  small  in  extent 


and  the  carcase, 
if  otherwise 
healthy,shall  not 


lesions   shall  be 
seized. 

"  In  view  of  the  greater  tendency  to  generalisation  of  tuberculosis 
in  the  pig,  we  consider  that  the  presence  of  tubercular  deposit  in 
any  degree  should  involve  seizure  of  the  whole  carcase  and  of  the 
organs. 

"In  respect  of  foreign  dead  meat,  seizure  shall  ensue  in  every 
case  where  the  pleura  have  been  '  stripped/  " 

The  tubercle  bacilli  are  most  easily  found  in  the  glands.  They 
are  scarce  in  the  caseating  nodules.  In  the  pig  it  is  difficult 
to  detect  the  bacilli  as  a  rule.  The  Eoyal  Commission  on 
Tuberculosis  emphasised  the  absence  of  bacilli  in  the  meat 
substance: — "In  tissues  which  go  to  form  the  butcher's  joint, 
the  material  of  tubercle  is  not  often  found  even  where  the 
organs  (lungs,  liver,  spleen,  membranes,  etc.)  exhibit  very  advanced 
or  generalised  tuberculosis;  indeed,  in  muscle  and  muscle  juice 
it  is  very  seldom  that  tubercle  bacilli  are  to  be  met  with; 
perhaps  they  are  somewhat  more  often  to  be  discovered  in  bone, 
or  in  some  small  lymphatic  gland  embedded  in  intermuscular  fat."  * 
The  chief  way  in  which  such  meat  substance  becomes  infected  with 
tubercle  appears  to  be  through  carelessness  of  the  butcher,  who 
perchance  smears  the  meat  substance  with  a  knife  that  has  been 
used  in  cutting  the  organs,  and  so  has  become  contaminated  with 
infected  material.  Very  instructive  also  are  the  results  at  which 
Dr  Sims  Woodhead  arrived  in  furnishing  evidence  for  the  same 
Commission  on  the  effect  of  cooking  upon  tuberculous  meat: — 
"Ordinary  cooking,  such  as  boiling  and  more  especially  roasting, 
though  quite  sufficient  to  sterilise  the  surface,  and  even  the  substance 
for  a  short  distance  from  the  surface  of  a  joint,  cannot  be  relied 
upon  to  sterilise  tubercular  material  included  in  the  centre  of  rolls 
of  meat,  especially  when  these  are  more  than  three  pounds  or  four 
pounds  weight.  The  least  reliable  method  of  cooking  for  this 
purpose  is  roasting  before  a  fire;  next  comes  roasting  in  an  oven, 
and  then  boiling."  f  From  this  statement  it  will  be  understood  that 

*  Royal  Commission  on  Tuberculosis,  Report,  1895,  part  i.,  p.  13. 
f  Ibid.,  p.  18. 


272  BACTERIA  IN  OTHER  FOODS 

rolled  meat  may  be  a  source  of  infection  to  a  greater  degree  than 
the  ordinary  fresh  joint,  and  this  is  borne  out  by  the  experience 
derived  from  epidemics  due  to  meat-poisoning. 

Tuberculous  meat  also  finds  its  way  occasionally  into  sausages, 
and  other  similarly  prepared  meat  foods. 

Swine  Fever  is  not  an  uncommon  disease  of  pigs,  and  makes  the 
meat  unfit  for  food.  Schutz  and  others  have  isolated  a  bacillus  from 
this  disease.  The  chief  post-mortem  signs  are  the  red  punctiform 
rash,  generally  becoming  confluent  on  the  back,  extremities,  and 
ears ;  the  ulceration  of  the  intestine,  and  the  characteristic  mottling 
of  the  lymph  glands. 

Anthrax  (see  p.  315),  Actinomycosis  (see  p.  321),  and  other  condi- 
tions are  described  elsewhere.  Many  parasitic  diseases  also  make  meat 
unfit  for  food. 

3.  Ice-cream 

In  1894  Dr  Klein  had  occasion  bacteriologically  to  examine  ice- 
cream sold  in  the  streets  of  London.  In  all  six  samples  were 
analysed,  and  in  each  sample  the  conclusions  resulting  were  of  a 
nature  sufficiently  serious  to  support  the  view  that  the  bacterial 
flora  was  not  inferior  to  ordinary  sewage.  The  water  in  which  the 
ice-cream  glasses  were  washed  was  also  examined,  and  found  to 
contain  large  numbers  of  bacteria. 

Since  that  date  many  investigations  have  been  made  into  ice- 
cream. It  appears  that  this  luxury  is  frequently  manufactured 
under  extremely  objectionable  circumstances,  and  with  anything  but 
sterilised  appliances.  Little  wonder,  then,  that  the  numbers  of 
bacteria  present  run  into  millions  per  c.c.  (varying  from  two  to 
twenty  millions  or  more).  In  nearly  all  recorded  cases,  the  quality 
of  the  germs  as  well  as  the  quantity  has  been  of  a  nature  to 
cause  some  concern.  B.  c'oli  communis  has  been  very  commonly 
found,  and  in  considerable  abundance.  The  Proteus  family,  which 
also  possesses  a  putrefactive  function,  is  common  in  ice-creams. 
The  common  water  bacteria  are  nearly  always  present.  B.  typliosus 
itself,  it  is  said,  has  been  isolated  from  some  ice-cream  which 
was  held  responsible  for  an  outbreak  of  enteric  fever.  The 
material  had  become  infected  during  process  of  manufacture  in  the 
house  of  a  person  suffering  from  unnotified  typhoid  fever. 

The  Manufacture  of  Ice-cream. — There  are,  practically  speaking,  three  methods 
of  manufacture : — 

(1)  The  real  ice-cream,  which  cannot  be  sold  at  a  low  price,  and  which  is  made 
simply  by  mixing  cream  (with  a  small  proportion  of  milk),  fruit  or  fruit  pulp  and 
sugar.     This  mixture  is  then  at  once  frozen. 

(2)  Milk  is  flavoured  with  fruit,  or  fruit  essence  and  sugar,  and  has  then  added 
to  it  a  small  quantity  of  dissolved  gelatine,  and  at  once  frozen.     In  these  two 
processes  there  is  no  boiling,  and  both  are  frozen  immediately  after  mixture. 


MANUFACTURE  OF  ICE-CREAM  273 

(3)  Skimmed  milk  is  boiled,  either  with  or  without  the  addition  of  some  form 
of  starch  (generally  corn  flour)  with  a  certain  number  of  eggs,  sugar  and  flavouring. 
Practically,  the  number  of  eggs  used  varies  inversely  with  the  amount  of  starch,  the 
effect  of  both  being  to  thicken  the  mixture,  and  in  some  cases  the  practice  has  been 
to  at  once  freeze  the  mixture  ;  in  others,  in  order  to  economise  the  consumption  of 
ice,  the  heated  liquid  has  been  allowed  to  cool  naturally.  Usually  the  number  of 
eggs  is  three  or  four  to  the  quart.  The  whole  is  again  boiled  (for  perhaps 
twenty  minutes),  after  which  it  is  set  aside  to  cool  until  the  following  morning, 
when  it  is  placed  in  the  "freezers"  and  frozen.  In  short,  the  mixture  contains 
no  cream,  and  is,  in  fact,  a  frozen  custard.  The  milk  and  eggs  are  generally 
obtained  locally,  and  are  usually  of  good  quality.  It  is  stated  that  if  such 
were  not  the  case  the  ice-cream  would  be  unpalatable,  owing  to  ill-flavour, 
and  therefore  unsaleable.  The  eggs  used  to  be  blown,  but  of  late  years 
that  practice  has  fallen  into  disuse,  and  they  are  now  broken  and  mixed  in  the 
ordinary  way.  The  boiling  is  carried  out  in  various  utensils  over  the  open  fire.  The 
mixture  stands  for  cooling  purposes  in  the  living  room,  or  in  any  out-of-the-way 
corner,  sometimes  in  the  open  yard  or  area.  The  freezing  takes  place  early  on  the 
following  morning  in  the  "  freezers."  A  "  freezer  "  consists  of  a  tin  or  galvanised  iron 
cylinder  or  container,  in  which  the  mixture  is  placed.  The  cylinder  fits  into  an 
outer  vessel  of  wood  or  metal  so  loosely  as  to  leave  an  inch  or  two  of  space  all 
round.  In  this  space  is  placed  broken  ice  and  salt,  and  the  inner  cylinder  is  rotated 
from  time  to  time  in  this  ice  medium.  No  ice  is  added  directly  or  indirectly  to  the 
mixture  itself,  nor  are  colouring  agents  used  as  a  rule.  The  utensils  and  materials 
appear,  as  a  general  rule,  to  be  clean. 

From  this  description,  which  applies,  generally  speaking,  to  the  street  ice-cream 
industry  in  London,  it  will  be  seen  that  the  "  ice-cream  "  is  boiled  for  some  time,  and 
in  all  probability  sterilised,  and  in  due  course  it  undergoes,  at  least  approximate, 
freezing.  These  facts,  added  to  the  generally  wholesome  condition  of  the  elementary 
materials  used,  would  appear,  at  first  sight,  to  place  the  substance  beyond  risk  of 
contamination.  But  the  critical  period  is  the  time  of  exposure  between  boiling  and 
freezing.  Boiling  sterilises,  but  freezing  does  not  sterilise.  Hence,  if  in  the  long 
cooling  process  the  substance  is  exposed  to  contaminated  surroundings,  the  result 
may  be,  in  effect,  a  contaminated  ice-cream.  Such,  in  fact,  frequently  occurs.* 
Hence  it  would  appear  that  it  is  not  the  process  of  manufacture  that  needs  super- 
vision so  much  as  the  general  condition  of  the  houses  in  which  the  substance  is 
made,  and  of  the  persons  who  make  it,  and  the  manufacture  so  far  as  length  of  time 
between  boiling  and  freezing  is  concerned. 

The  important  stage  of  the  operation  is,  therefore,  that  between  the  boiling  and 
freezing.  Attention  has  been  drawn  to  the  fact  that  the  majority  of  the  specific 
pathogenic  bacilli  discovered  in  ice-cream  are  of  a  non-sporing  variety,  and  that, 
therefore,  if  originally  present  in  the  material,  would  have  been  destroyed  by  boil- 
ing, and,  if  found  subsequently,  must  have  gained  access  to  the  material  while 
cooling.  The  subsequent  freezing,  while  it  might  inhibit  such  bacilli,  would 
certainly  not  destroy  them,  and  on  ingestion  and  melting  their  growth  and  develop- 
ment would  again  commence. 

Some  dozen  outbreaks  of  disease  have  been  attributed  to  the 
consumption  of  ice-creams.  A  typhoid  epidemic  occurred  in 
Liverpool  (27  cases)  in  1897  due  to  ice-cream,  and  an  earlier  epidemic 
of  the  same  disease  traceable  to  the  same  cause  occurred  at  Deptford 
in  1891  (Turner).  Kecently,  a  small  outbreak  occurred  in  the  city 
of  London  affecting  16  telegraph  boys.  The  symptoms  were  colic 
and  diffuse  abdominal  pains,  headache,  vomiting,  diarrhoea,  and 
nervous  depression.  Dr  Collingridge's  inquiry  resulted  in  the 
following  conclusions : — 

*  See  investigations  by  Klein,  Cook,  Wilkinson,  Foulerton,  and  others. 

S 


274  BACTERIA  IN  OTHER  FOODS 

(1)  That  in  a  number  of  cases  of  illness  occurring  among  young 
persons  of  a  susceptible  age,  the  symptoms  were  strictly  identical, 
and  were  characteristic  of  poisoning  by  ingestion  of  toxic  material. 

(2)  That  the  cases  reported  followed  the  ingestion  of  ice-creams. 

(3)  That  ice-creams  subsequently  obtained  at  shops  frequented 
by  the  patients  contained  bacilli  of  a  virulent  character. 

(4)  That  the  symptoms  observed  were  those  generally  following 
the  ingestion  of  material  containing  such  bacilli. 

(5)  That  where  pathogenic  bacilli  were  found,  the  ices  had  been 
manufactured   under  insanitary  conditions.      The  majority  of  the 
manufacturers  are  aliens,  and  although  the  premises  may  be  kept  in 
a  fairly  sanitary  condition,  their  personal  habits  unfortunately  leave 
much  to  be  desired  where  the  preparation  of  food  is  concerned. 

Dr  Klein  examined  24  samples  of  ice-cream  from  the  same 
locality,  and  found  13  (or  54  per  cent.)  to  be  poisonous  to  guinea- 
pigs.*  The  writer  traced  18  cases  of  typhoid  fever  in  1902  to  the 
consumption  of  contaminated  ice-cream.-)-  Owing  to  outbreaks  of 
this  nature  the  London  County  Council  (General  Powers)  Act,  1902 
(sects.  42-45),  has  given  powers  for  controlling  this  trade : — 

(a)  Ice-cream  must  be  made  and  stored  in  sanitary  premises. 

(b)  It  must  not  be  made  or  stored  in  living  rooms. 

(c)  Strict  precautions  must  be  taken  as  to  protection  from  con- 
tamination. 

(d)  Cases  of  infectious  disease  must  be  reported. 

(e)  The  name  and  address  of  the  maker  must  appear  on  street 
barrows. 

These  regulations  are  new  for  London,  though  they  have  practi- 
cally been  in  existence  in  Glasgow  since  1895,  and  in  Liverpool  since 
1898. 

It  should  not  be  forgotten  that  ice-cream  may  have  deleterious 
effects  on  the  consumer  owing  to  its  low  temperature  or  to  the 
presence  of  alkaloidal  poisons  of  the  nature  of  tyro-toxicon  which 
have  been  detected  in  such  substance  as  well  as  in  milk  (Mount 
Morgan  outbreak)  and  cheese  (Michigan  and  Finsbury  outbreaks). 

Ice  contains  bacteria  in  varying  quantities,  from  20  per  c.c.  to 
10,000  or  more.  Nor  is  variation  in  number  affected  alone  by  the 
conditions  of  the  water,  for  samples  collected  from  one  and  the  same 
place  differ  widely.  The  quality  follows  in  large  measure  the 
standard  of  the  water. 

Water  bacteria,  B.  coli,  putrefactive  and  even  pathogenic  bacteria, 
have  been  found  in  ice.  Many  organisms  can  live  without  much 
difficulty,  and  are  most  numerous  in  ice  containing  air-bubbles. 

*  Report  of  Medical  Officer  of  City  of  London,  1902,  pp.  116-26. 
t  Report  on  Health  of  Finsbury,  1902,  p.  67. 


ICE  275 

Dr  Prudden,  of  New  York,  performed  a  series  of  experiments  in 
1887  to  show  the  relative  behaviour  of  bacteria  in  ice.  Taking  half 
a  dozen  species,  he  inoculated  sterilised  water  and  reduced  it  to  a 
very  low  temperature  for  a  hundred  and  three  days,  with  the 
following  results : — B.  prodigiosus  diminished  from  6300  per  c.c.  to 
3000  within  the  first  four  days,  to  22  in  thirty-seven  days,  and 
vanished  altogether  in  fifty-one  days;  a  liquefying  water  bacillus 
numbering  800,000  per  c.c.  at  the  commencement,  had  disappeared 
in  four  days ;  Staphylococcus  pyogenes  aureus  and  B.  fluorescens  showed 
large  numbers  present  at  the  end  of  sixty-six  and  seventy-seven  days 
respectively ;  B.  typhosus,  which  was  present  1,000,000  per  c.c.  after 
eleven  days,  fell  to  72,000  after  seventy-seven  days,  and  7000  at  the 
end  of  one  hundred  and  three  days.  Anthrax  bacilli  are  susceptible  to 
freezing,  but  their  spores  are  practically  unaffected  (Frankland).  From 
these  facts  it  will  be  seen  that  bacteria  live,  but  do  not  multiply,  in  ice. 

Hutchings  and  Wheeler  recently  examined  some  ice  suspected  of 
conveying  typhoid  fever  at  the  St  Lawrence  State  Hospital  on  the 
river  St  Lawrence.  The  fragments  were  melted  in  a  clean  vessel  at 
room  temperature,  after  which  a  considerable  black  sediment  deposited 
itself  in  the  vessel.  Cultures  and  plates  were  made  in  the  usual 
way,  and  B.  coli  and  B.  typliosus  were  both  isolated.*  The  last- 
named  had  the  following  characters  : — On  nutrient  agar  it  grew 
readily,  in  broth  growth  without  pellicle,  in  lactose  media  no  fermenta- 
tion occurred,  on  potato  the  "  invisible  "  growth,  litmus  milk  became 
alkaline  without  coagulation,  and  the  bacillus  was  morphologically 
identical  with  B.  typhosus.  With  the  serum  of  typhoid  patients 
characteristic  agglutination  occurred.  Blumer  found  the  number 
of  bacteria  in  some  of  the  same  ice  was  30,400  per  c.c.  (agar)  and 
50,400  per  c.c.  (gelatine).  Many  colon  bacilli  were  present. 

Sedgwick  and  Winslow  have  also  carefully  studied  the  influence 
of  natural  and  normal  conditions  of  cold  upon  the  typhoid  bacillus 
in  particular.  The  experiments  were  carried  out  with  special  refer- 
ence to  the  danger  of  conveyance  of  the  disease  in  question  by 
polluted  ice,  and  with  reference  to  the  seasonal  distribution  of  the 
disease.  The  matter  was  undoubtedly  one  that  called  for  investiga- 
tion, and  notably  so  in  America  where  ice  and  iced  drinks  are  in 
such  universal  demand. 

The  apparent  purity  of  ice  is  deceptive.  It  is  true  that  water  in 
freezing  undergoes  a  certain  amount  of  purification.  It  loses,  on 
conversion  into  ice,  saline  constituents,  contained  air,  and  a  certain 
proportion  of  organic  suspended  matter.  At  the  same  time,  it  is  not 
entirely  freed  from  microbes.  The  figures  quoted  by  Sedgwick  and 
Winslow  show  that  snow-ice  may  contain  an  average  of  more  than 
600  bacteria  per  cubic  centimetre. 

*  American  Jour,  of  Medical  Sciences,  Oct.  1903,  p.  683. 


276  BACTERIA  IN  OTHER  FOODS 

Laboratory,  experiments  have  confirmed  the  conclusion  that 
a  freezing  process  is  not  necessarily  fatal  to  bacterial  life 
(see  p.  18).  We  have  instances  of  bacteria  multiplying  at  zero, 
and  of  their  survival  after  six  months'  exposure  to  the  temperature 
of  liquid  air.  It  would  appear  that  about  90  per  cent,  of  the 
ordinary  water  bacteria  are  eliminated  by  the  process  of  freezing. 
In  the  case  of  a  specific  pathogenic  organism  such  as  the 
B.  typhosus,  less  than  1  per  cent,  survive  simple  freezing  for  a 
period  of  fourteen  days.  Complete  sterility  does  not  occur  even 
at  the  end  of  three  months,  whilst  a  process  of  alternate  thawing 
and  freezing,  if  on  the  whole  more  fatal  to  the  typhoid  germs 
than  a  simple  freezing,  is  equally  unsuccessful  in  effecting  an 
absolute  sterilisation  of  the  infected  water.  The  reduction  in 
the  number  of  typhoid  bacilli  in  chilled  water  is  approximately 
as  great  as  occurs  in  ice.  Cold  exercises  an  inhibitory  action 
as  regards  the  typhoid  bacillus,  and  in  natural  ice  there  is  a 
supplementary  purifying  influence  to  be  taken  into  account,  as,  at 
the  time  of  freezing,  90  per  cent,  of  the  germs  are  thrown  out  by  a 
process  of  physical  exclusion.  Therefore,  the  danger  of  infection  in 
the  case  of  ice,  if  it  is  minimised,  is  not  abolished.  A  certain  number 
of  typhoid  bacilli  do  remain  alive,  and  these  may,  on  rethawing, 
undergo  a  rapid  multiplication  outside  as  well  as  inside  the  human 
body.  And  it  has  likewise  to  be  remembered  that  it  is  notoriously 
difficult  to  trace  the  exact  channels  of  infection  in  sporadic  cases  of 
typhoid  fever.  Sedgwick  and  Winslow  have  rightly  drawn  attention 
to  the  unfavourable  conditions  furnished  by  natural  ice  for  the 
propagation  of  the  typhoid  organism. 

In  making  a  bacterial  investigation  into  the  flora  of  ice  and  ice- 
cream, it  is  necessary  to  remember  that  considerable  dilution  with 
sterilised  water  is  required.  The  usual  methods  of  examining  water 
and  milk  are  adopted. 

4.  Bread 

Bread  forms  an  excellent  medium  for  moulds,  but  unless 
specially  exposed  the  bacteria  in  it  are  few.  Waldo  and  Walsh 
have,  however,  demonstrated  that  baking  does  not  sterilise  the 
interior  of  bread.  These  observers  cultivated  numerous  bacteria 
from  the  centre  of  newly-baked  London  loaves.*  The  writer  has 
recently  made  a  series  of  examinations  of  the  air  of  some  nine  or  ten 
underground  bakehouses  in  central  London.  The  general  result  of 
these  investigations  was  that  the  air  of  the  typical  underground 
bakehouses  examined — (1)  contained  14'8  volumes  per  10,000  of 
carbonic  acid  gas,  C02  (as  compared  with  4'9  in  above-ground  bake- 
houses, and  4'3  in  the  street) ;  (2)  that  it  contained  between  10  and 
*  Brit.  Med.  Jour.,  1895,  vol.  ii.,  p.  519. 


BREAD  277 

24  per  cent,  less  moisture  than  outside  air  surrounding  the  bake- 
houses ;  and  (3)  that  it  contained  at  least  four  times  more  bacteria 
than  surrounding  street  air,  and  three  times  more  bacteria  than  the 
air  of  a  typical  above-ground  bakehouse.*  (See  also  p.  86.) 

The  normal  fermentation  of  bread  with  which  all  bakers  are 
familiar  is  due  to  the  energy  of  the  yeast  plant  growing  in  the 
dough.  Any  other  fermentation  going  on  at  the  same  time  as  the 
normal  one,  or  arising  after  the  bread  has  left  the  oven,  must  be 
looked  upon  as  abnormal. 

Flour  or  dough  is  open  to  infection  by  bacteria,  and  scrupulous 
cleanliness  is  absolutely  necessary  to  avoid  unfavourable  fermenta- 
tions. Bacteria  are  especially  numerous  in  low-grade  flours ;  in  fact, 
the  poorer  the  flour  the  larger  the  number  of  injurious  organisms. 
Prescott  has  lately  shown  that  flour  may  contain  bacilli  indistinguish- 
able from  the  B.  coli.  This  organism  is  more  liable  to  be  found 
in  poor  than  in  high-class  flours. 

1.  Sour  Bread. — The  commonest  abnormal  fermentation  of  bread 
produces  what  is  known  as  "sour  bread,"  which  means  that  the 
odour  and  flavour  of  the  bread  are  "  sour  "  to  the  senses  of  smell  and 
taste.  Lactic  and  butyric  germs  are  commonly  found  in  poor  flours, 
where  they  remain  in  a  dormant  condition  until  provided  with  the 
essentials  necessary  for  their  growth — moisture,  a  sufficient  tempera- 
ture, and  proper  and  adequate  food  supply.  The  food  supply 
naturally  surrounds  them,  and  when  water  is  added  to  the  flour, 
and  the  temperature  is  raised  to  between  70°  and  90°  F.,  they  are 
able  to  reproduce  and  rapidly  manifest  their  presence  by  the  products 
they  form.  Dough,  with  considerable  moisture  present,  or,  as  it  is 
termed,  "  slack,"  gives  bacteria  a  better  environment,  and  consequently 
sourness  is  more  apt  to  increase  rapidly  in  such  doughs. 

Acid-producing  germs  are  also  present  in  many  samples  of 
yeast.  Analyses  of  a  large  number  of  yeast  samples  used  for 
bread-making  purposes,  have  shown  that  many  of  them  contain 
injurious  bacteria  which  may  lessen  the  alcoholic  fermentation. 
If,  on  the  other  hand,  the  normal  alcoholic  fermentation  is  at 
first  vigorous,  and  then  diminishes,  it  gives  bacteria  opportunity  to 
grow.  Hence,  overproved  dough  is  especially  liable  to  become  sour. 
Dirty  utensils,  tubs  or  troughs,  harbour  injurious  bacteria  which  are 
able  to  reproduce  when  given  favourable  conditions.  All  cracks  and 
crevices  which  harbour  food  are  teeming  with  life,  usually  undesirable 
from  the  bakers'  standpoint,  and,  therefore,  absolute  cleanliness 
should  be  the  rule  in  every  detail. 

Acetic  bacteria,  which  are  often  present  in  flours,  sometimes 
cause  trouble,  and  as  these  bacteria  require  a  plentiful  supply  of 
oxygen,  it  has  been  suggested  that  all  dough  should  be  kept  as  much 

*  Special  Report  on  Bakehouses  in  Finsbury,  1902. 


278  BACTERIA  IN  OTHER  FOODS 

as  possible  out  of  contact  with  the  air.  It  is  doubtful  if  such  a 
remedy  is  practical,  as  the  lowering  of  the  temperature  follows  the 
removal  of  covers  on  the  dough  troughs  and  retards  the  whole  course 
of  fermentation. 

2.  Sticky,  Slimy,  or  Viscous  Bread. — This  affection  is  not  nearly 
as  common  as  the  preceding,  yet  the  number  of  cases  recorded  is 
quite  large,  and  this  abnormal  fermentation  is  frequently  met  with 
in  country  districts.     As  the  name  implies,  the  bread,  usually  the 
crumb  near  the  centre  of  the  loaf,  is  slimy  or  sticky.     The  stringiness 
increases   with  age,  a  proof  of  the  living  nature   of   the   trouble. 
Cases  of  sticky  bread  usually  occur  in  the  warm  summer  months, 
the  high  temperature  favouring  the  growth  of  the  bacteria  which  pro- 
duce the  trouble.     From  this  sticky  bread  it  is  comparatively  easy  to 
isolate  an  organism  which,  when  placed  in  sterilised  bread,  is  able 
to  produce  the  stickiness  met  with  under  natural  conditions,  thus 
proving  the  relation  of  bacteria  to  the  trouble.     The  specific  germ 
causing  stickiness,  known  as  the  "potato  bacillus"  on  account  of 
the  frequency  with  which  it  is  met  with  on  potatoes,  is  also  formed 
in  yeast  cake.     Harrison  has  repeatedly  found  this  germ  present  in 
both  dried  and  compressed  yeast  cake.     Given  favourable  conditions 
for  rapid  growth,  this  organism  might  produce  epidemics  of  slimy 
bread  at  any  time.     The  bacillus  forms  spores  able  to  resist  un- 
favourable conditions.     This  germ  is  occasionally  met  with  in  milk. 
Slimy  bread  may  be  controlled  by  exercising  absolute  cleanliness 
in  the  yeast  tubs  and  kneading  troughs,  and  by  the  proper  sterilisa- 
tion of  the  brew  or  ferment  by  the  use  of  a  certain  quantity  of  hops. 
In  a  number  of  experiments  made  with  hop  extracts  it  has  been 
found  that  even  a  small  quantity  of  good  hops  (one  half -ounce  to 
the  gallon)  has  some  antiseptic  power  and  hinders  the  development 
of  the  potato  bacillus,  without  injuring  the  activity  of  the  yeast. 
The   bread  should  be  kept  in  a  cool  place  after  baking,  for  this 
stickiness  is  most  prevalent  during  hot  weather,  and  a  cool  tempera- 
ture prevents  the  rapid  growth  of  the  organism. 

3.  Musty  or  Mouldy  Bread. — Musty  or  mouldy  bread  is, .  as  a 
rule,  only  met  with  after  the  bread  has  been  cut  and  allowed  to 
stand  several  days.     Occasionally,  however,  we  find  bread  only  one 
day  old  affected  with  mustiness.     The  specific  organism  is  the  mould 
Mucor  mucedo,  which  has  action  on  bread,  producing  a  musty  odour 
without  decomposing  the  bread.     But  the  chemical  composition  of 
the   bread   is   changed   by  the  growth  of  mould,  and  this  change 
favours  the  subsequent  growth  of  any  bacteria  that  may  be  present. 
Flours  which  have  become  damp,  or  even  very  low-grade  flours,  may 
have  this  mould  present  in  large  amount,  and  although  the  organism 
is  killed  by  the  baking  process,  yet  the  musty  flavour  persists  and  is 
present  in  the  baked  loaf. 


WATERCRESS,  ETC.  279 

4.  Red  or  "  Bloody "  Bread. — Bloody,  or  red  bread,  is  not  an 
affection  which  often  troubles  bakers,  but  it  sometimes  makes  its 
appearance  in  the  household.  The  microbe  which  produces  this 
affection  is  of  great  historical  interest.  Livy  refers  to  its  occurrence 
in  the  Eoman  army,  and  it  is  said  to  have  appeared  during  the  siege 
of  Troy.  There  are  various  records  of  its  occurrence  in  England 
during  the  Middle  Ages,  and  early  in  the  nineteenth  century  a  large 
quantity  of  red  spotted  bread  occurred  in  the  province  of  Padua,  in 
North  Italy.  It  is  possibly  due  to  B.  prodigiosus  or  other  similar 
chromogenic  organism,  and  is  traceable  to  contamination  of  the 
bread. 

5.  Miscellaneous  Foods 

Watercress  has  frequently  been  found  to  be  the  vehicle  of  bacteria 
if  grown  in  polluted  water.  There  are  several  instances  on  record 
where  the  consumption  of  such  contaminated  watercress  has  caused 
disease.  In  June  and  July  1903  an  outbreak  of  enteric  fever 
occurred  in  Hackney,  in  N.E.  London,  in  which  there  were  110 
cases  of  the  disease,  of  whom  55'5  per  cent,  had  consumed  watercress 
which  was  shown  to  have  been  grown  in  polluted  water.  The  latter 
contained  50  B.  coli  per  c.c.,  and  the  cresses  themselves  were  markedly 
contaminated  with  sewage  organisms  of  intestinal  type.  Altogether, 
17  samples  of  watercress  were  examined,  and  every  one  of  them 
revealed  the  bacteria  of  sewage.  This  was  a  fairly  clear  case  of 
conveyance  of  enteric  infection,  as  (1)  the  excess  of  enteric  fever 
corresponded  with  the  season  for  watercress,  viz.,  June  to  September ; 
(2)  the  excess  of  cases  of  enteric  fever  was  amongst  watercress 
eaters,  viz.,  55  per  cent,  for  the  whole  period ;  (3)  watercress  eaters 
suffered  more  than  three  times  as  much  as  non-watercress  eaters, 
who  constituted  only  %7'5  per  cent,  of  the  entire  population;  (4) 
samples  of  watercress,  taken  from  the  places  where  infected  persons 
market,  were  found  on  bacteriological  examination,  to  be  sewage- 
polluted  ;  and,  (5)  a  large  proportion  of  the  polluted  samples  were 
found  to  be  cultivated  in  beds  fed  by  almost  undiluted  sewage.* 

Other  foods  and  leverages  (including  aerated  waters)  have  from 
time  to  time  been  contaminated  with  bacteria  to  the  injury  of  the 
consumer,  but  the  above  represent  the  chief  foods  infected.  Sausages 
have  frequently  been  found  to  be  contaminated.  In  Liverpool, 
Boyce  found  B.  coli  present  in  all  17  samples  examined,  and  B. 
enteritidis  sporogenes  in  2  out  of  17.  Pork  pies,  tinned  meats  and  pastes, 
chicken,  jellies,  etc.,  have  been  shown  to  harbour  injurious  organisms.f 

*  Report  on  Outbreak  of  Enteric  Fever  at  Hackney,  1903  (Dr  King  Warry). 
f-  Report  on  Health  of  Liverpool,  1902,  p.  172. 


CHAPTER  IX 


BACTERIA  AND   DISEASE 

Growth  of  Knowledge  of  Bacteria  as  Disease  Producers — Channels  of  Infection — 
How  Bacteria  cause  Disease — Diphtheria :  Conditions  of  Infection — Scarlet 
Fever,  Typhoid  Fever,  Epidemic  Diarrhoea:  Conditions  of  Infection — 
Suppuration  and  Abscess  Formation — Anthrax — Pneumonia — Influenza — 
Actinomycosis— Glanders. 

PROBABLY  the  most  universally  known  fact  respecting  bacteria  is 
that  they  are  related  in  some  way  to  the  production  of  disease. 
Yet  we  have  seen  that  it  was  not  as  disease-producing  agents  that 
they  were  first  studied.  Indeed,  it  is  only  within  comparatively  the 
latest  period  of  the  two  centuries  during  which  they  have  been 
more  or  less  under  observation  that  our  knowledge  of  them  as 
causes  of  disease  has  assumed  any  exactitude  or  general  recognition. 
Nor  is  this  surprising,  for  although  an  intimate  relationship  between 
fermentation  and  disease  had  been  hinted  at  in  the  middle  of  the 
seventeenth  century,  it  was  not  till  the  time  of  Pasteur  that  the 
bacterial  cause  of  fermentation  was  experimentally,  and  finally, 
established. 

In  the  middle  of  the  seventeenth  century  men  learned,  through 
the  eyes  of  Leeuwenhoek,  that  drops  of  water  contained  "moving 
animalcules."  A  hundred  years  later  Spallanzani  demonstrated  the 
fact  that  decomposition  and  fermentation  were  set  up  in  boiled 
vegetable  infusions  when  outside  air  was  admitted,  but  when  it 
was  withheld  from  these  boiled  infusions  no  such  change  occurred. 
Almost  a  hundred  years  more  passed  before  the  epoch-making  work 
of  Tyndall  and  Pasteur,  who  separated  these  putrefactive  germs 
from  the  air.  Quickly  following  in  their  footsteps  came  Davaine 

280 


KOCH'S  POSTULATES  281 

and  Pollender,  who  found  in  the  blood  of  animals  suffering  from 
anthrax  the  now  well-known  specific  bacillus  of  that  disease. 
Improvements  in  the  microscope  and  in  methods  of  cultivation 
(Koch's  plate  method  in  particular)  soon  brought  an  army  of  zealous 
investigators  into  the  field,  and  during  the  last  thirty  years  one 
disease  after  another  has  been  traced  to  a  bacterial  origin.  We  may 
summarise  the  vast  collection  of  historical,  physiological,  and  patho- 
logical research  extending  from  1650  to  1904  in  three  great  periods : 
The  period  of  detection  of  living,  moving  cells  (Leeuwenhoek  and 
others  in  the  seventeenth  century) ;  the  period  of  the  discovery  of 
their  close  relationship  to  fermentation  and  putrefaction  (Spallanzani, 
Schulze,  Schwann,  in  the  eighteenth  century);  and,  thirdly,  the 
period  of  appreciation  of  the  role  of  bacteria  in  the  economy  of 
nature  and  in  the  production  of  disease  (Tyndall,  Pasteur,  Lister, 
Koch,  in  the  nineteenth). 

But  we  must  look  less  cursorily  at  the  growth  of  the  idea  of 
bacteria  as  the  cause  of  disease.  More  than  two  hundred  years  ago 
Eobert  Boyle  (1627-91),  the  philosopher  who  did  so  much  towards 
the  foundation  of  the  present  Eoyal  Society,  wrote  an  elaborate  treatise 
on  The  Pathological  Part  of  Physic.  He  was  one  of  the  earliest 
scientists  to  declare  that  a  relationship  existed  between  fermentation 
and  disease.  When  more  accurate  knowledge  was  attained  respecting 
fermentation,  great  advance  was  consequently  made  in  the  etiology 
of  disease.  The  preliminary  discoveries  of  Fuchs  and  others  between 
1840  and  1850  had  relation  to  the  existence  in  diseased  tissues  of 
a  large  number  of  bacteria.  But  this  was  no  proof  that  these  germs 
caused  disease.  It  was  not  till  Davaine  had  inoculated  healthy 
animals  with  bacilli  from  the  blood  of  an  anthrax  carcase,  and  had 
thus  reproduced  the  disease,  that  reliance  could  be  placed  upon  that 
bacillus  as  the  vera  causa  of  anthrax.  Too  much  emphasis  cannot 
be  laid  upon  the  idea,  that  unless  a  certain  organism  produces  in 
healthy  tissues  the  disease  in  question,  it  cannot  be  considered  as 
proven  that  the  particular  organism  is  related  to  the  disease  as 
cause  to  effect.  In  order  to  secure  a  standard  by  which  all  investi- 
gators should  test  their  results,  Koch  introduced  four  postulates. 
Until  each  of  the  four  has  been  fulfilled,  the  final  conclusion  respect- 
ing the  causal  agent  in  any  bacterial  disease  must  be  considered  sub 
judice.  The  postulates  are  as  follows : — 

(a)  The  organism  must  be  demonstrated  in  the  circulation   or 
tissues  of  the  diseased  animals. 

(b)  The   organism    thus    demonstrated    must   be   cultivated   in 
artificial  media  outside  the  body,  and  successive  generations  of  a 
pure  culture  of  that  organism  must  be  obtained. 

(c)  Such  pure  cultures  must,  when  introduced  into  a  healthy  and 
susceptible  animal,  produce  the  specific  disease. 


282  BACTERIA  AND  DISEASE 

(d)  The  organism  must  be  found  and  isolated  from  the  circulation 
or  tissues  of  the  inoculated  animal. 

It  is  evident  that  there  are  some  diseases — for  example,  cholera, 
leprosy,  and  typhoid  fever — which  are  not  communicable  to  lower 
animals,  and  therefore  their  virus  cannot  be  made  to  fulfil  postulate 
(c).  In  such  cases  there  is  no  choice.  They  cannot  be  classified 
along  with  tubercle  and  anthrax.  Bacteriologists  have  little  doubt 
that  Hansen's  bacillus  of  leprosy  is  the  cause  of  that  disease,  yet 
it  has  not  fulfilled  postulates  (b)  and  (c).  Nor  has  the  generally 
accepted  bacillus  of  typhoid  fever  fulfilled  postulate  (c),  yet  by  the 
majority  it  is  provisionally  accepted  as  the  agent  in  producing  the 
disease.  Hence  it  will  be  seen  that,  though  there  is  an  academical 
classification  of  causal  pathogenic  bacteria  according  as  they  respond 
to  Koch's  postulates,  yet  nevertheless  there  are  a  number  of  patho- 
genic bacteria  which  are  looked  upon  as  causes  of  disease  provisionally. 
The  bacilli  of  anthrax  and  tubercle,  with  perhaps  the  organisms  of 
suppuration,  tetanus,  plague,  and  actinomycosis,  stand  in  the  first 
order  of  pathogenic  germs.  Then  comes  a  group  awaiting  further 
confirmation,  which  includes  the  organisms  related  to  typhoid  fever, 
cholera,  malaria,  leprosy,  epidemic  diarrhoea,  and  pneumonia.  Then 
comes  in  a  third  category,  a  long  list  of  diseases,  such  as  scarlet 
fever,  small-pox,  measles,  rabies,  and  others  too  numerous  to  mention, 
in  which  the  nature  of  the  causal  agent  is  still  unknown.  Hence  it 
must  not  be  supposed  that  every  disease  has  its  germ,  and  without 
a  germ  there  is  no  disease.  Such  universal  assertions,  though  not 
uncommonly  heard,  are  devoid  of  accuracy. 

In  the  production  of  bacterial  disease  there  are  two  factors. 
First,  there  is  the  body  tissue  of  the  individual ;  secondly,  there  is 
the  specific  organism. 

Whatever  may  be  said  hereinafter  with  regard  to  the  power  of 
micro-organisms  to  cause  disease,  we  must  understand  one  cardinal 
point,  namely,  that  bacteria  are  never  more  than  causes,  for  the  nature 
of  disease  depends  upon  the  behaviour  of  the  organs  or  tissues  with 
which  the  bacteria  or  their  products  meet  (Yirchow).  Fortunately  for 
a  clear  conception  of  what  "  organs  and  tissues "  mean,  these  have 
been  reduced  to  a  common  denominator,  the  cell.  Every  living 
organism,  of  whatever  size  or  kind,  and  every  organ  and  tissue  in 
that  living  organism,  contains  and  consists  of  cells.  Further,  these 
cells  are  composed  of  organic  chemical  substances  which  are  not 
themselves  alive,  but  the  mechanical  arrangement  of  which  determines 
the  direction  and  power  of  their  organic  activity  and  of  their  resist- 
ance to  the  specific  agents  of  disease.  With  these  facts  clearly 
before  us,  we  may  hope  to  gain  some  insight  into  the  reasons  for 
departure  from  health. 

The  normal  living  tissues  have  an  inimical  effect  upon  bacteria. 


RESISTANCE  OF  THE  TISSUES  283 

Saprophytic  bacteria  of  various  kinds  are  normally  present  on 
exposed  surfaces  of  skin  or  mucous  membrane.  Tissues,  also,  which 
are  dead  or  depressed  in  vitality  from  injury  or  previous  disease, 
but  which  are  still  in  contact  with  the  living  body,  afford  an  excellent 
nidus  for  the  growth  of  bacteria.  Still  these  have  not  the  power, 
unless  specific,  to  thrive  in  the  normal  living  tissue.  It  has  been 
definitely  shown  that  the  natural  fluids  of  the  body  have  in  their 
fresh  state  protective  substances  (alexines)  which  prevent  bacteria 
from  flourishing  in  these  tissues.  Such  protection  depends  in  measure 
upon  the  number  of  invading  germs  as  well  as  their  quality,  for  the 
killing  power  of  blood  and  lymph  must  be  limited.  Buchner  has 
pointed  out  that  the  antagonistic  action  of  these  fluids  depends  in  part 
possibly  upon  phagocytosis,  but  largely  upon  a  chemical  condition 
of  the  serum.  The  blood,  then,  is  no  friend  to  intruding  bacteria. 
Its  efforts  are  to  a  certain  extent  seconded  by  the  lymphoid  tissue 
throughout  the  body.  Rings  of  lymphoid  tissue  surround  the  oral 
openings  of  the  trachea  and  oesophagus,  and  the  tonsils  are  masses 
of  lymphoid  tissue.  Composed  as  it  is  of  cells  having  a  germicidal 
influence  when  in  health,  the  lymphoid  tissue  may  afford  formidable 
obstruction  to  invading  germs. 

All  the  foregoing  points  in  one  direction,  namely,  that  if  the 
tissues  are  maintained  in  sound  health,  they  form  a  very  resistant 
barrier  against  disease-producing  germs.  But  we  know  from  experi- 
ence that  a  full  measure  of  health  is  not  often  the  happy  condition 
of  human  tissues.  There  are  a  variety  of  circumstances  which 
predispose  the  individual  to  disease.  One  of  the  commonest  forms 
of  predisposition  is  that  due  to  heredity.  Probably  it  is  true  that 
what  are  known  as  "hereditary  diseases"  are  due  far  more  to  a 
hereditary  predisposition  than  to  any  transmission  of  the  virus  itself 
in  any  form.  Again,  antecedent  disease  predisposes  the  tissues  to 
form  a  nidus  for  bacteria,  and  conditions  of  environment  or  personal 
habits  act  powerfully  in  the  same  way.  Damp  soils  must  be  held 
responsible  for  many  disasters  to  health,  not  directly,  but  indirectly, 
by  predisposition ;  dirty  houses  and  insanitary  houses,  dusty  trades 
and  injurious  occupations,  have  a  similar  effect.  Any  one  of  these 
different  influences  may  in  a  variety  of  ways  affect  the  tissues  and 
increase  their  susceptibility  to  disease.  Not  infrequently  we  may 
get  them  combined.  For  example,  the  following  is  not  an  unlikely 
series  of  events  terminating  in  consumption  (tuberculosis  of  the 
lungs): — (a)  The  individual  is  predisposed  by  inheritance  to 
tuberculosis;  (b)  an  ordinary  chronic  catarrh,  which  lowers  the 
resisting  power  of  the  lungs,  may  be  contracted ;  (c)  the  epithelial 
collections  in  the  air  vesicles  of  the  lung — i.e.  dead  matter  attached 
to  the  body — afford  an  excellent  nidus  for  bacteria;  (d)  owing  to 
occupation,  or  personal  habits,  or  surroundings,  the  patient  comes 


284  BACTERIA  AND  DISEASE 

within  a  range  of  tubercular  infection,  and  the  specific  bacilli  of 
tubercle  gain  access  to  the  lungs.  The  result  will  be  a  case  of 
consumption  more  or  less  acute  according  to  environment  and 
treatment. 

Channels  of  Infection 

The  channels  of  infection  by  which  organisms  gaiii  the  vantage- 
ground  afforded  by  the  depressed  tissues  are  various,  and  next  to 
the  maintenance  of  resistant  tissues  they  call  for  most  attention 
from  the  physician  and  surgeon.  It  is  in  this  field  of  preventive 
medicine — that  is  to  say,  preventing  infective  matter  from  entering 
the  tissues  at  all — that  science  has  triumphed  in  recent  years.  It 
is,  in  short,  applied  bacteriology. 

1.  Pure  Heredity. — This  term  is*  to  be  understood  in  this  connec- 
tion as  concerned  with  actual  transmission  of  germs  of  disease  from 
the  mother  to  the  child  in  utero.     That  such  conveyance  may  occur 
is  admitted,  but  it  is  certainly  not  frequent,  nor  is  bacterial  disease 
widely  spread  by  this  means.    The  transmission  of  tendency  (diathesis) 
is,  of  course,  another  matter,  and  there  can  be  little  doubt  that  ante- 
natal conditions  exert  an  influence  on  bacterial  diseases  of  infancy. 

2.  Inoculation,  or  inserting  virus  directly  through  a  broken  sur- 
face of  skin,  is  a  method  of  producing  diseases  in  animals  commonly 
used  in  experimental  work.     Such  inoculations  may  be  subcutaneous, 
intravenous,  intracerebral,  intraperitoneal,  etc.     In  the  natural  pro- 
duction of  disease,  inoculation  is  also  a  not  uncommon  channel  of 
infection.      Injuries   of   the   skin   caused    by  instruments,   gunshot 
wounds,  broken  glass  or  china,  etc.,  may  serve  as  the  point  of  intro- 
duction of  specific  virus.     Tetanus  is  commonly  an  inoculated  disease. 
Malaria  must  now  also  be  so  considered.     Local  tuberculosis  is  not 
infrequently  produced  by  inoculation  through  a  broken  skin  surface. 

3.  Contagion  indicates  that  a  disease  is  transmitted  by  personal 
contact,  through  unbroken  skin  surfaces.     Small-pox,  measles,  ring- 
worm, and  other  diseases  may  be  thus  contracted.     It  is  not  unlikely 
that  as  our  knowledge  grows,  the  diseases  to  be  defined  as  spread  by 
contagion  will  become  less. 

4.  The  Alimentary  System. — Many  diseases  are  spread    by  the 
consumption  of  infected  food  or  water,  and  in  children  the  sucking 
of  dirty  objects  may  iiitroduce  germs  of  disease  into  the  alimentary 
canal.      Milk,  cream,   butter,   cheese,   ice-cream,   oysters,   shell-fish, 
meats  of  various  kinds,  vegetables,  water-cress,  ice,  and  a  large  variety 
of  foods,  have  been  the  means  of  introducing  pathogenic  organisms 
into  the  body,  and  in  this  way  enteric  fever,  cholera,  dysentery,  and  a 
large  number  of  acute  and  chronic  diseases  are  originated.     Water- 
borne  disease  furnishes  a  large  percentage  of  such  cases. 

5.  The  Respiratory   Tract. — The  air  may  become  infected  with 


ACTION  OF  BACTERIA  285 

pathogenic  organisms,  which  may  be  inhaled,  and  thus  gain  entrance 
to  the  body  and  set  up  disease.  Diphtheria  and  pulmonary  tubercu- 
losis are  two  examples.  In  this  channel  of  infection  pathogenic 
bacteria  must,  as  a  rule,  be  present  in  large  numbers,  or  must  meet 
with  devitalised  and  non-resisting  tissues,  to  set  up  disease. 

These,  then,  are  the  five  possible  ways  in  which  germs  gain 
access  to  the  body  tissues.  The  question  now  arises,  How  do 
bacteria,  having  obtained  entrance,  set  up  the  process  of  disease  ?  For 
a  long  time  pathologists  looked  upon  the  action  of  these  microscopic 
parasites  in  the  body  as  similar  to,  if  not  identical  with,  the  larger 
parasites  sometimes  infesting  the  human  body.  Their  work  was 
viewed  as  a  devouring  of  the  tissues  of  the  body.  Now  it  is  well 
known  that,  however  much  or  little  of  this  may  be  done,  the 
specific  action  of  pathogenic  bacteria  is  of  a  different  nature.  It  is 
twofold.  We  have  the  action  of  the  bacteria  themselves,  and  also 
of  their  products  or  toxins.  In  particular  diseases,  now  one  and 
now  the  other  property  comes  to  the  front.  In  bacterial  diseases 
affecting  or  being  transmitted  mostly  by  the  blood,  it  is  the  toxins 
which  act  chiefly.  The  convenient  term  infection  is  applied  to  those 
conditions  in  which  there  has  been  a  multiplication  of  living 
organisms  after  they  have  entered  the  body,  the  word  intoxication 
indicating  a  condition  of  poisoning  brought  about  by  their  products. 
It  will  be  apparent  at  once  that  we  may  have  both  these  conditions 
present,  the  former  before  the  latter,  and  the  latter  following  as  a 
direct  effect  of  the  former.  Until  intoxication  occurs,  there  may  be 
few  or  no  symptoms;  but  directly  enough  bacteria  are  present  to 
produce  in  the  body  certain  poisons  in  sufficient  amount  to  result  in 
more  or  less  marked  tissue  change,  then  the  symptoms  of  that  tissue 
change  appear.  This  period  of  latency  between  infection  and  the 
appearance  of  the  disease  is  known  as  the  incubation  period.  Take 
typhoid,  for  example.  A  man  drinks  a  typhoid-polluted  water. 
For  about  fourteen  days  the  bacilli  are  making  headway  in  his  body 
without  his  being  aware  of  it.  But  at  the  end  of  that  incubation 
period  the  signs  of  the  disease  assert  themselves.  Professor  Watson 
Cheyne  and  others  have  maintained  that  there  is  some  exact 
proportion  between  the  number  of  bacteria  gaining  entrance  and  the 
length  of  the  incubation  period. 

Speaking  generally,  we  may  note  that  pathogenic  bacteria  divide 
themselves  into  two  groups :  those  which,  on  entering  the  body, 
pass  at  once,  by  the  lymph  or  blood-stream,  to  all  parts  of  the  body, 
and  become  more  and  more  diffused  throughout  the  blood  and 
tissues,  although  in  some  cases  they  settle  down  in  some  spot 
remote  from  the  point  of  entrance,  and  produce  their  chief  lesions 
there.  Tubercle  and  anthrax  would  be  types  of  this  group.  On 
the  other  hand,  there  is  a  second  group,  which  remain  almost 


286  BACTERIA  AND  DISEASE 

absolutely  local,  producing  only  little  reaction  around  them,  rarely 
passing  through  the  body  generally,  and  yet  influencing  the  whole 
body  eventually  by  means  of  their  ferments  or  toxins.  Of  such,  the 
best  representatives  are  tetanus  and  diphtheria.  The  local  site  of 
the  bacteria  is,  in  this  case,  the  local  factory  of  the  disease. 

Whilst  the  mere  bodily  presence  of  bacteria  may  have  mechanical 
influence  injurious  to  the  tissues  (as  in  the  small  peripheral 
capillaries  in  anthrax),  or  may  in  some  way  act  as  a  foreign  body 
and  be  a  focus  of  inflammation  (as  in  tubercle),  the  real  disease- 
producing  action  of  pathogenic  bacteria  depends  upon  the  chemical 
poisons  (toxins)  formed  directly  or  indirectly  by  them.  Though 
within  recent  years  a  great  deal  of  knowledge  has  been  acquired 
about  the  formation  of  these  bodies,  their  exact  nature  is  not  at 
present  known.  They  are  allied  to  the  proteoses,  and  are  frequently 
described  as  tox-albumens.  It  may  be  found,  after  all,  that  they 
are  not  of  a  proteid  nature.  Sidney  Martin  has  pointed  out  that 
there  is  much  that  is  analogous  between  the  production  of  toxins 
and  the  production  of  the  final  bodies  of  digestion.  Just  as  ferments 
are  necessary  in  the  intestine  to  bring  about  a  change  in  the  food 
by  which  the  non-soluble  albumens  shall  be  made  into  soluble 
peptones,  and  thus  become  absorbed  through  the  intestinal  wall,  so 
also  a  ferment  may  be  necessary  to  the  production  of  toxins.  Such 
ferments  have  not  as  yet  been  isolated,  but  their  existence  in 
diphtheria  and  tetanus  is,  as  we  have  seen,  extremely  likely. 
However  that  may  be,  it  is  now  more  or  less  established  that 
there  are  two  kinds  of  toxic  bodies,  differing  from  each  other  in 
their  resistance  to  heat.  It  may  be  that  the  one  most  easily 
destroyed  by  heat  is  a  ferment  and  possibly  an  originator  of  the 
other.  A  second  division  which  has  been  suggested  for  toxic  bodies, 
and  to  which  reference  will  be  made,  is  intracellular  and  extra- 
cellular, according  to  whether  or  not  the  poison  exists  within  or 
without  the  body  of  the  bacillus. 

Lastly,  we  may  turn  to  consider  the  action  of  the  toxins  on  the 
individual  in  whose  body-fluids  they  are  formed.  It  is  hardly 
necessary  to  say  that  any  action  which  bacteria  or  toxins  may  have 
will  depend  upon  their  virulence,  in  some  measure  upon  their 
number,  and  not  a  little  upon  the  channel  of  infection  by  which 
they  have  gained  entrance.  It  could  not  be  otherwise.  If  the 
virulence  is  attenuated,  or  if  the  invasion  very  limited  in  numbers, 
it  stands  to  reason  that  the  pathogenic  effects  will  be  correspondingly 
small  or  absent.  The  influence  of  the  toxins  is  twofold.  In  the 
first  place,  (i.)  they  act  locally  upon  the  tissues  at  the  site  of  their 
formation,  or  at  distant  points  by  absorption.  There  is  inflamma- 
tion with  marked  cell-proliferation,  and  this  is,  more  or  less  rapidly, 
followed  by  a  specific  cell-poisoning.  The  former  change  may  be 


ACTION  OF  BACTERIA  287 

accompanied  by  exudation,  and  simulate  the  early  stages  of  abscess 
formation ;  the  latter  is  the  specific  effect,  and  results,  as  in  leprosy 
and  tubercle,  in  infective  nodules.  The  site  in  some  diseases,  like 
typhoid  (intestinal  ulceration,  splenic  and  mesenteric  change)  or 
diphtheria  (membrane  in  the  throat),  may  be  definite  and  always  the 
same.  But,  on  the  other  hand,  the  site  may  depend  upon  the  point 
of  entrance,  as  in  tetanus.  The  distant  effects  of  the  toxin  are  due 
to  absorption,  but  what  controls  its  action  it  is  impossible  to  say. 
We  only  know  that  we  do  find  pathological  conditions  in  certain 
organs  at  a  distance  from  the  site  of  disease,  and  without  the 
presence  of  bacteria.  We  have  a  parallel  in  the  action  of  drugs ; 
for  example,  a  drug  may  be  given  by  the  mouth,  and  yet  produce  a 
rash  in  some  distant  part  of  the  body.  In  the  second  place,  (ii.) 
toxins  produce  toxic  symptoms.  Fever  and  many  of  the  nervous 
conditions  resulting  from  bacterial  action  must  thus  be  classified. 
We  have,  it  is  true,  the  physical  signs  of  the  pathological  tissue 
change,  for  example,  the  large  spleen  of  anthrax  or  the  obstruction 
from  diphtheritic  membrane.  But,  in  addition  to  these,  we  have 
general  symptoms,  as  fever,  in  which  after  death  no  tissue  change 
can  be  found. 

We  may  now  consider  briefly  some  of  the  more  important  forms 
of  disease  produced  by  bacteria.* 

Diphtheria 

Diphtheria  is  an  infective  disease  characterised  by  a  variety 
of  clinical  symptoms,  including  a  severe  inflammation  usually 
followed  by  a  fibrinous  infiltration  (constituting  a  membrane)  of 
certain  parts.  The  membrane  ultimately  breaks  down.  The  parts 
affected  are  the  mucous  membrane  of  the  fauces,  larynx,  pharynx, 
trachea,  and  sometimes  wounds,  or  the  inner  wall  of  the  stomach. 
Diphtheritic  conjunctivitis  may  also  occur.  The  common  sign 
of  the  disease  is  the  membrane  in  the  throat;  but  muscle 

*  Bacterial  diseases  may  be  classified  as  follows  : — 

(1)  Diseases  common  to  man  and  certain  animals,  and  presumably  trans- 

missible from  animals  to  man,  and  vice  versa,  e.g.  bubonic  plague  and 
tuberculosis. 

(2)  Diseases  common  to  man  and  animals,  but  not  known  to  be  directly 

transmissible,  e.g.  actinomycosis,  tetanus.  Diphtheria,  belongs  to  this 
class,  or  Group  (1)  or  (5). 

(3)  Diseases  transmitted  from  animals  to  man,  but  not  as  a  rule  communicated 

from  man  to  man  owing  to  interfering  conditions,  e.g.  anthrax,  glanders, 
rabies,  vaccinia,  foot-and-mouth  disease,  meat-poisoning,  psittacosis, 
and  possibly  infections  due  to  pus  bacteria. 

(4)  Certain  specific  symbiotic  relations  requiring  two  hosts  for  the  complete 

cycle  of  life  of  the  micro-organisms,  e.g.  malaria,  trichinosis,  tape-worm 
infection. 

(5)  Diseases  occurring  in  man,  but  not,  as  far  as  known,  in  animals,  e.g. 

typhoid  fever,  gonorrhoea,  leprosy. 


288  BACTERIA  AND  DISEASE 

weakness,  syncope,  albuminuria,  post-diphtheritic  paralyses,  con- 
vulsions, and  many  other  symptoms  guide  the  physician  in 
diagnosis  and  the  course  of  the  disease.  It  begins  as  a  local 
disease,  and  the  greyish-white  membranous  deposit,  already  referred 
to,  is  produced.  The  toxins  or  poisons  resulting  from  the  growth 
and  multiplication  of  the  bacillus  are  absorbed  into  the  blood  stream, 
and  general  symptoms  follow.  The  incubation  period  is  from  two 
to  seven  days. 

Although  diphtheria  owes  its  name  to  the  false  membrane  seen 
in  the  throats  of  typical  cases,  it  is  now  almost  universally  recognised 
that  in  many  cases  of  undoubted  diphtheria  no  membrane  is  formed. 
The  occurrence  of  a  nasal  form  of  diphtheria  has,  too,  in  recent  years 
been  recognised,  and  as  such  cases  are  not  easily  recognisable  without 
a  bacteriological  examination,  they  are  very  liable  to  remain  undi- 
agnosed  and  be  left  free  to  spread  the  infection. 

Thefons  et  origo  of  the  disease  is  the  specific  bacillus.  Without 
the  presence  of  that  organism  it  is  not  possible  to  have  diphtheria. 
Yet  that  organism  may  exist  in  the  healthy  throat  without  producing 
the  recognised  clinical  symptoms  of  diphtheria.  It  may  be  conveyed 
to  the  human  throat  in  a  variety  of  ways,  for  example,  by  kissing 
and  other  forms  of  contact,  or  by  drinking  milk  and  other  con- 
taminated foods.  In  a  perfectly  healthy  throat  it  may  do  no  mischief. 
But  in  a  sore  throat  or  in  the  throat  of  a  weakly  person,  it  might 
readily  set  up  severe  and  even  fatal  disease.  Anything,  therefore, 
which  tends  to  lower  the  vitality  of  the  individual  may  play  an 
important  part  in  propagating  diphtheria,  and  must  be  as  carefully 
considered  as  any  agency  which  might  directly  or  indirectly  introduce 
the  bacillus  to  the  human  throat.  Some  epidemics  have  been  due 
to  school  influence;  other  epidemics  have  been  brought  about 
through  an  infected  milk  supply ;  and  yet  other  outbreaks  are  due  to 
the  introduction  of  a  case  of  diphtheria  into  a  susceptible  community, 
weakened  by  insanitary  surroundings  or  the  prevalence  of  previous 
sore  throat. 

Further,  there  is  reason  to  suppose  that  Bacillus  diphtherias  may 
retain  its  virulence — and  possibly  spend  a  stage  of  its  cycle  of 
existence  as  a  saprophyte — in  the  soil,  in  dust,  and  even  in  the 
throat  for  months.  Three  or  four  weeks  is  the  average  length  of 
time  for  its  presence  in  the  throat,  but,  as  a  matter  of  fact,  all  the 
conditions  in  the" throat — mucous  membrane,  blood-heat,  moisture,  and 
air — are  extremely  favourable  to  the  bacillus,  and  it  may  linger 
there  far  beyond  the  time  of  disappearance  of  clinical  symptoms  of 
the  disease. 

The  Bacillus  diphtherias  was  isolated  from  the  many  bacteria 
found  in  the  membrane  by  Loffler  (1884).  Klebs  had  previously 
identified  the  bacillus  as  the  cause  of  the  disease  (1883).  It  is  a 


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[To  face  page  288. 


Of  THE 

VNiYSRSfTY 

OF 


DIPHTHERIA  280 

slender  rod,  straight  or  slightly  curved,  and  remarkable  for  its 
beaded  appearance ;  there  are  also  irregular  and  club-shaped  forms. 
It  differs  in  size  according  to  its  culture 
medium,  but  is  generally  3  or  4  /UL  in 
length.  Cobbett  and  Graham-Smith  re- 
cognise five  morphological  types  of  diph- 
theria bacilli  on  young  serum  cultures : 
— (1)  Oval  bacilli,  with  one  unstained 
septum ;  (2)  long,  faintly-stained,  irregu- 
larly-beaded bacilli;  (3)  long,  regularly- 
beaded  bacilli — "  streptococcal "  forms ; 
(4)  segmented  bacilli ;  and  (5)  uniformly- 
stained  bacilli.  All  these  forms  are 
longer  than  the  pseudo-diphtheria  bacilli, 
more  curved,  and  generally  with  clubbed  FIG.  24.— Diagram  of  sadiius 
ends.  Their  arrangement  to  each  other 

is   generally  likened    to    Chinese    characters.      In    the    membrane 
which  is   its   strictly  local   habitat  in  the  body — indeed,  with  the 
exception  of  the  secretions  of  the  pharynx  and  larynx,  and  occasion- 
ally in  lymph-glands  and  the  spleen,  the  bacillus  is  found  practically 
nowhere  else  in  the  body — it  sometimes  shows   parallel  grouping, 
lies   on    the    surface    of    the    exudation,  and    is    separated    from 
the   mucous   membrane   by   the   fibrin.      It   is   mixed   with   other 
organisms,   which   are   performing  their  part  in  complicating  the 
disease,  or   are   normal  inhabitants   of   the   mouth.     The   bacillus 
possesses   five   negative   characters:   namely,  it   has  no  spores,  no- 
threads,  and  no  power  of  motility ;  it  does  not  liquefy  gelatine;  nor 
does  it  produce  gas.     It  is  stained  with  Loffler's  methylene  blue,  and 
shows  metachromatic  granules  and  polar  staining.     It  is  generally 
best  to  use  the  stain  in  dilute  form.     The  favourable  temperature  is 
blood-heat,  though  it  will  grow  at  room  temperature.     It  is  aerobic, 
and,  indeed,  prefers  a  current  of  air.     Loffler  contrived  a  medium 
for  cultivation  which  has  proved  most  successful.     It  is  made  by 
mixing  three  parts  of  ox-blood  serum  with  one  part  of  broth  contain- 
ing 1  per  cent,  of  glucose,  1  per  cent,  of  peptone,  and  \  per  cent,  of 
common   salt;   the   whole   is   coagulated.     Upon   this  medium  the 
Klebs-Loffler  bacillus  grows  rapidly  in  eighteen  or  twenty  hours, 
producing   scattered,  "nucleated,"  round,  white  colonies,  becoming 
yellowish.     Horse  serum  is  used  by  some  bacteriologists  instead  of 
ox  serum.     Lorrain  Smith   and   Marmorek   also   devised   excellent 
serum    media.      The    bacillus   grows    well   in    broth,    but   without 
producing    either   a   pellicle"^  or    turbidity ;    it   can   grow    on    the 
ordinary   media,    though    its    growth    on    potato    is    not    readily 
visible;  on  the  white  of  egg   it   flourishes   extremely   well.      In   a 
moist   condition,   as   a    rule,    the    bacillus    has   a    low    degree    of 

T 


290  BACTERIA  AND  DISEASE 

resistance,  but  when  in  a   dry  condition  it  can   survive   for   long 
periods. 

The  bacillus  is  secured  for  diagnostic  purposes  by  one  of  two 
methods :  (a)  Either  a  piece  of  the  membrane  is  detached,  and  after 
washing  carefully  examined  by  culture  as  well  as  the  microscope ; 
or  (b)  a  "  swab  "  is  made  from  the  infected  throat,  cultured  on  serum, 
and  incubated  at  37°  C.  for  eighteen  hours,  and  then  microscopically 
examined.  Both  methods — and  there  is  no  further  choice — present 
some  difficulties  owing  to  the  large  number  of  bacteria  found  in  the 
throat.  Hence  a  negative  result  must  be  accepted  with  reserve. 
Indeed  the  rule  to  follow  is  three  examinations  before  deciding  that 
a  throat  is  free  from  infectivity,  and  it  should  be  remembered  that 
about  20  per  cent,  of  all  cases  of  diphtheria  offer  no  bacteriological 
evidence  of  infection.*  It  therefore  comes  back  to  the  point  of 
broad  judgment  and  common-sense.  The  clinical  condition  is  the 
main  fact  for  guidance,  and  the  bacteriological  must  not  usurp 

itt 

Locally,  the  bacillus  produces  inflammatory  change  with  fibrinous 
exudation  and  some  cellular  necrosis.  In  the  membrane  a  ferment 
is  probably  produced  which,  unlike  the  localised  bacilli,  passes 
throughout  the  body,  and.  by  digestion  of  the  proteids,  produces 
albumoses  and  an  organic  acid  which  have  the  toxic  influence.  The 
toxins  act  on  the  blood-vessels,  the  nerves,  the  muscle  fibres  of  the 
heart  (hyaline  degeneration  and  even  fatty  changes),  and  many  of  the 
more  highly  specialised  cells  of  the  body.  Thus  we  get  degenerative 
changes  in  the  kidney  (cloudy  swelling,  and,  clinically,  albuminuria) 
in  cells  of  the  central  nervous  system,  in  the  peripheral  nerves 
(post -diphtheritic  paralysis),  and  elsewhere,  these  pathological  condi- 
tions setting  up,  in  addition  to  the  membrane,  the  symptoms  of  the 
disease.  The  bacillus  is  pathogenic  for  the  horse,  ox,  rabbit,  guinea- 
pig,  cat,  and  some  birds.  Cases  are  on  record  of  supposed  infection 
of  children  by  cats  suffering  from  the  disease.  Eoux  and  Yersin 
in  1888-89  showed  that  the  diphtheria  bacillus  is  capable  of  pro- 
ducing the  various  phenomena  associated  with  the  disease,  including 
the  formation  of  false  membrane  and  diphtheritic  paralysis.  They 
also  succeeded  in  separating  and  studying  the  toxin,  which  they 
found  to  be  capable  of  producing  all  the  effects  produced  by  the 
bacillus.  In  1890  appeared  the  great  work  by  Behring,  to  which 
reference  will  be  made  subsequently ;  and  the  observations  in  regard 
to  diphtheria  made  in  that  work  were  extended  and  strengthened  in 
a  paper  by  Behring  and  Wernicke  in  1892.  At  the  Medical  Congress 
at  Buda-Pesth  in  1894,  Roux  read  a  paper  on  the  treatment  of  diph- 
theria by  diphtheria  antitoxin,  which  first  proved  to  the  medical 

*  Jour.  ofHyyiene,  1903,  p.  217. 

t  See  also  Brit.  Med.  Jour.,  1900,  ii.,  p.  907  (Andrewes). 


DIPHTHERIA  291 

world  that  this  was  the  one  method  of  successfully  combating  the 
disease.  The  experimental  and  clinical  data,  and  the  favourable 
statistics  brought  forward  by  Roux,  at  once  put  this  method  in  a 
secure  position  from  the  practical  standpoint.* 

Conditions   Favourable   to   the   Spread    of    Diphtheria.— 

Sir  Eichard  Thome  Thome  held  that  soil,  and  especially  surface  soil, 
when  considered  in  connection  with  relative  altitude,  slope,  aspect, 
and  prevailing  winds,  plays  a  part  in  the  maintenance  and  diffusion 
of  diphtheria,  and  possibly  has  relation  with  its  beginnings.  He 
believed  that  where  a  surface  soil  retained  moisture  and  organic 
refuse,  and  where  buildings  founded  on  such  soil  were  exposed  to 
cold  wet  winds,  there  you  had  conditions  likely  to  foster  diphtheria.f 
Dr  Newsholme  of  Brighton  considers  that  such  conditions  act  as 
"vital  depressants,"  favouring  sore  throat  and  catarrh,  and  thus 
preparing  the  way  for  the  inroads  of  the  diphtheria  bacillus.  He 
concludes  as  the  result  of  extended  investigations  that  "  diphtheria 
only  becomes  epidemic  in  years  in  which  the  rainfall  is  deficient,  and 
the  epidemics  are  on  the  largest  scale  when  three  or  more  years  of 
deficient  rainfall  immediately  follow  each  other.  Occasionally,  dry 
years  are  unassociated  with  epidemic  diphtheria,  though  usually  in 
these  instances  there  is  evidence  of  some  rise  in  the  curve  of 
diphtheritic  death-rate.  Conversely,  diphtheria  is  nearly  always  at 
a  very  low  ebb  during  years  of  excessive  rainfall,  and  is  only 
epidemic  during  such  years  when  the  disease  in  the  immediately 
preceding  dry  years  has  obtained  a  firm  hold  of  the  community  and 
continues  to  spread  presumably  by  personal  infection."  J  Newsholme 
thinks  the  specific  micro-organism  has  a  double  cycle  of  existence : 
one  phase  passed  in  the  soil,  saprophytic ;  another  in  the  human 
organism,  parasitic. 

Insanitary  surroundings  necessarily  act  prejudicially.  Damp  and 
ill-constructed  houses  and  bad  drainage,  have  undoubtedly  played  a 
part,  and  that  not  a  small  part,  in  diphtheria  outbreaks.  The 
position  of  many  houses  must  inevitably  lead  to  dampness ;  there  is 
also  the  dampness  arising  from  undrained  and  unpaved  curtilages ; 
and  lastly,  there  is  damp  and  steamy  atmosphere.  Now  these  con- 
ditions cannot  but  affect  the  health  of  children  and  give  rise  to  sore 
throats  and  similar  complaints.  When  to  this  dampness  of  houses 
is  added  the  pollution  of  the  soil,  the  undrained  condition  of  towns, 
and  the  nuisances  readily  arising  from  ash-pits,  cess-pits,  and  similar 
methods  of  refuse  removal  in  close  proximity  to  the  houses,  there  is 

*  See   also    Brit.   Med.  Jour.,  1900,  it,  pp.  658-62  (Marsden) ;    Metropolitan 
Asylums  Board  Reports,  1869-1902.     See  also  pp.  425-431  of  present  volume. 
f  The  Natural  History  of  Diphtheria,  p.  17. 
£  Epidemic  Diphtheria  (Newsholme),  p.  157. 


292  BACTERIA  AND  DISEASE 

ample  ground  for  concluding  that  the  sanitary  circumstances  of  a 
town  may  be  such  as  to  depress  the  physical  vitality  of  children,  and 
lessen  their  powers  of  resistance  to  infectious  disease  once  introduced 
among  them.  Thus  the  insanitary  conditions  named  weaken  the 
physique  of  the  children,  as  well  as  preparing  favourable  external 
circumstances  for  the  growth  and  multiplication  of  the  germs  of 
disease.  Hence  it  must  come  about  that  from  time  to  time  a  disease 
like  diphtheria  will  take  on  an  increased  virulence  as  well  as  a  higher 
measure  of  epidemicity. 

But  general  conditions  do  not  wholly  account  for  the  occurrence 
of  diphtheria.  Apart  from  these  general  conditions  personal  infection 
is  the  chief  means  by  which  diphtheria  is  spread. 

Infection  has  been  proved  to  be  conveyed  by  nasal  discharge  of 
infected  persons,  or  by  kissing  infected  persons,  or  by  sucking  sweets, 
pencils,  pens,  slates,  and  other  articles  in  schools.  School  influence 
as  an  agency  in  the  dissemination  of  diphtheria  was  shown  as  far 
back  as  1876  by  Mr  W.  H.  Power,  and  since  that  date  abundant  con- 
firmatory evidence  has  been  forthcoming.  In  1894  Mr  Shirley 
Murphy,  medical  officer  to  the  London  County  Council,  reported 
that  there  had  been  an  increase  in  diphtheria  mortality  in  London 
at  school  ages  (three  to  ten)  as  compared  with  other  ages  since  the 
Elementary  Education  Act  became  operative  in  1871 ;  that  the 
increased  mortality  from  diphtheria  in  populous  districts,  as  com- 
pared with  rural  districts,  since  1871,  might  be  due  to  the  greater  effect 
of  the  Education  Act  in  the  former ;  and  that  there  was  a  diminution 
of  diphtheria  in  London  during  the  summer  holidays  at  the  schools 
in  1893,  but  that  1892  did  not  show  any  marked  changes  for  August. 
In  1896  Professor  W.  R  Smith,  the  medical  officer  to  the  London 
School  Board,  furnished  a  report*  on  this  same  subject  of  school 
influence,  in  which  he  produced  evidence  to  show  that  the  recru- 
descence of  the  disease  in  1881-90  was  greatest  in  England  and 
Wales  at  the  age  of  two  to  three  years,  and  in  London  at  the  age  of 
one  to  two  years,  in  both  cases  before  school  age;  that  age  as  an 
absolute  factor  in  the  incidence  of  the  disease  is  enormously  more 
active  than  any  school  influence,  and  that  personal  contact  is  another 
important  source  of  infection. 

Although  it  is  said  that  "statistics  can  be  made  to  prove 
anything,"  there  can  be  little  doubt  that  both  of  these  reports 
contain  a  great  deal  of  truth ;  nor  are  these  truths  wholly  incompatible 
with  each  other.  They  both  emphasise  age  as  a  great  factor  in  the 
incidence  of  the  disease,  and  whatever  affects  the  health  of  the  child, 
population,  like  schools,  must  play,  directly  or  indirectly,  a  not 
unimportant  part  in  the  transmission  of  the  disease. 

*  Jour,  of  State  Med.,  1896,  vol.  iv.,  p.  169;  see  also  L.C.C.  Education  lleport, 
1904,  No.  718. 


DIPHTHERIA  293 

Infection  can  also  be  conveyed  by  means  of  milk  (see  p.  211). 
Clothing  and  other  articles  which  have  been  in  contact  with  infected 
persons  may  carry  the  bacillus.  Birds  and  cats  also  have  been,  as 
far  as  can  be  judged,  channels  of  infection  (Klein,  Bruce  Low,  arid 
others).  But  there  is  from  a  bacteriological  point  of  view  another 
body  of  facts  altogether  which  affect  the  spread  of  the  disease,  namely, 
the  behaviour  of  the  bacillus  in  the  throat. 

The  Diphtheria  Bacillus  in  the  Human  Throat.— Since  1896 
it  has  been  known  that  the  diphtheria  bacillus  may  remain  for  long 
periods  in  the  throat. 

"The  persistence  of  the  diphtheria  bacillus  for  periods  up  to  eight 
weeks  is  of  very  common  occurrence  whether  antitoxin  be  given  or 
not;  indeed,  the  majority  of  cases  appear  to  retain  bacilli  in  the 
throat  for  from  two  to  nine  weeks."  *  After  the  ninth  week,  the 
number  falls  off  very  rapidly,  but  not  infrequently  the  bacillus 
remains  in  the  throat  for  100  days,  and  it  has  been  known  to 
remain  more  than  200  days.  This  persistence  of  diphtheria  bacilli 
in  the  throat  may  play  an  important  part  in  determining  the  spread 
of  the  disease  by  means  of  such  cases  which  are  supposed  to  be  no 
longer  infective.  "For  it  is  now  a  matter  of  common  experience 
that  so  long  as  these  diphtheria  bacilli,  even  the  less  virulent  forms, 
remain  in  the  crypts  of  the  tonsils,  etc.,  so  long  is  the  patient  a 
centre  of  infection,  the  diphtheria  bacilli  present  resuming,  under 
favourable  conditions,  their  more  virulent  form"  (Woodhead).  In 
this  way  diphtheria  bacilli  can  be  readily  transmitted  by  patients 
who  are  apparently  no  longer  suffering  from  the  effects  of  the  disease, 
to  those  who  have  weak  or  ulcerated  throats.  In  precisely  a  similar 
manner,  may  the  bacillus  be  conveyed  to  articles  of  attire  and  articles 
of  food,  such  as  milk  (as  at  Leeds  in  1903). 

Further,  whilst  in  72  per  cent,  of  notified  definite  cases  of 
diphtheria  the  bacillus  may  be  found,  it  has  been  shown  that  in 
apparently  healthy  persons  who  have  not  suffered  from  diphtheria, 
the  B.  diphtherias  may  be  present.  Loftier  found  diphtheria  bacilli  in 
the  throats  of  4  out  of  160  healthy  children,  and  Park  and  Beebe 
found  similar  virulent  bacilli  in  8  out  of  330  "healthy"  throats. 
Hewlett  and  Murray  found  15  per  cent,  of  the  children  in  a  general 
hospital  had  diphtheria  bacilli  in  their  throats.")*  Kober  J  examined 
diphtheria  cultures  from  two  series  of  healthy  persons.  The  first 
series  comprised  128  individuals  known  to  have  been  in  recent  contact 
with  patients  suffering  from  diphtheria.  The  diphtheria  bacillus  was 

*  Report  on  the  Bacteriological  Diagnosis  and  Antitoxic  Serum  Treatment  of  Cases 
admitted  to  the  Hospitals  of  the  Metropolitan  Asylums  Board,  1895-96,  by  Professor 
Sims  Woodhead,  sect.  2,  1902,  pp.  14,  28,  31. 

f  Brit.  Med.  Jour.,  1901,  vol.  i.,  p.  1474. 

%  Revue  des  Maladies  de  VEnfance,,  Juillet,  1900. 


294  BACTERIA  AND  DISEASE 

found  in  the  throat  of  8  per  cent,  of  these.  The  second  series  com- 
prised 600  individuals  who  had  not  recently  come  into  contact  with 
any  diphtheria  cases — from  5  of  these  a  bacillus  similar  to  diphtheria 
was  isolated.  It  was  rather  short,  with  swollen  ends,  and  was  not 
pathogenic  to  guinea-pigs.  Denny  of  Brooklyn  examined  235 
healthy  persons,  and  found  the  diphtheria  bacillus  in  one  case.  Biggs 
met  with  32  cases  out  of  330  healthy  persons,  and  the  Committee  of 
the  Massachusetts  Board  of  Health  reported  that  1-2  per  cent,  of 
healthy  persons  amongst  the  general  public  are  infected  with  diph- 
theria bacilli.  Only  17  per  cent,  of  such  bacilli  appear,  however, 
to  be  virulent.  Goadby  examined  100  healthy  school  children,  and 
found  18  with  diphtheria  bacilli,  but  the  disease  was  prevalent  in 
the  neighbourhood.* 

It  is  certain  that,  as  a  rule,  "  healthy "  throats  do  not  yield  the 
true  B.  diphtheria  unless  those  examined  have  been  in  contact  with 
infected  persons.  But  that  raises  the  real  difficulty  in  practical 
public  health  work.  The  definitely  diseased  and  the  definitely 
healthy  persons  can  be  arranged  for.  It  is  the  apparently  healthy 
person,  who  coming  into  contact  with  the  infected  person  acts  as  a 
"  carrier "  of  infection,  that  creates  the  problem.  The  actual  per- 
centage of  such  persons  varies  widely.  In  infected  families  it  may 
be  50  per  cent.  (Park  and  Beebe),  or  100  per  cent.  (Goadby).  In 
schools  and  institutions  the  percentage  is,  of  course,  lower.  Goadby 
found  it  to  be  34  per  cent,  in  the  Poplar  Union  Schools,  but  it  may  be 
as  low  as  7  (Thomas).  Aaser  found  that  19  per  cent,  of  the  "  contacts  " 
in  a  soldiers'  barracks  contained  the  bacillus  in  their  throats,  and 
Denny  found  in  a  truant  school  that  the  percentage  was  11.  In 
hospital  wards  it  is  commonly  above  20,  and  among  the  general 
public  above  10  per  cent.f  These  figures  at  once  explain  the  spread 
of  diphtheria.  They  also  suggest  the  methods  of  prevention. 

In  the  ordinary  diphtheria  epidemic,  whether  large  or  small,  these 
methods  are  mainly  five.  First,  the  actual  cases  of  diphtheria  must 
be  effectually  and  promptly  isolated.  Secondly,  the  throats  of  contact 
persons  should  be  bacteriologically  examined,  and  those  persons  in 
whose  throats  the  bacillus  is  found — "  carriers  " — should  be  isolated, 
and  their  throats  treated.  Thirdly,  sore  throats  in  the  immediate 
vicinity  of  the  diphtheria  infection  should  be  similarly  examined. 
Fourthly,  thorough  disinfection  should  take  place  in  respect  of  in- 
fected houses,  and  inquiry  made  as  to  school  influences,  social  habits, 
etc.,  of  infected  households.  Fifthly,  antitoxin  should  be  used  as 
prophylactic  in  infected  families. 

The  new  facts  respecting  the  persistence  of  the  bacillus  in  the 

*  See  Jour,  of  Hygiene,  1903  (Graham-Smith),  p.  216  ;  and  1904,  p.  255. 
t  See  Jour,  of  Hygiene,  1904,  pp.    258-328  (Graham-Smith) ;    and  Practitioner, 
1903,  vol.  ii.,  pp.  715-21  (Newman). 


PSEUDO-DIPHTHERIA  BACILLUS  295 

throat  indicate  the  importance  of  throat  treatment  which  there  has 
been  a  tendency  to  ignore  on  account  of  the  increased  use  of  antitoxin. 
But  antitoxin  has  little  direct  effect  upon  the  bacilli  in  the  throat, 
which  should,  therefore,  be  treated  by  painting  with  perchloride  of 
mercury  (1-500),  or  washed  with  chlorine  water  or  permanganate  of 
potash  (1-300).  The  methods  to  adopt  in  order  to  clean  the  throat 
of  the  diphtheria  bacillus  are  three,  namely,  (a)  complete  isolation  of 
the  patient,  coupled  with  open-air  treatment;  (b)  application  of 
antiseptics  to  throat;  and  (e)  antitoxin. 

The  Pseudo-diphtheria  Bacillus.* — In  this  group  should  not 
be  included  non-virulent  forms  of  the  diphtheria  bacillus,  but  allied 
forms.f  Loftier  and  Hofmann  described  a  bacillus  having  similar 
morphological  characters  as  the  true  B.  diphtheria;,  except  that  it  had 
no  virulence.  It  is  frequently  found  in  healthy  throats,  and  is 
believed  by  some  to  be  a  common  inhabitant  of  the  mouths  of  the 
poorer  classes,  especially  children.  The  chief  differences  between  the 
real  and  the  pseudo-bacillus  are — 

1.  The  pseudo-bacillus  is  thicker  in  the  middle  than  at  the  poles, 
and  not  so  variable  as  the  B.  diphtherice.     Polar  staining  is  generally 
less  marked.     It  appears  as  an  oval  bacillus  of  variable  length,  gener- 
ally having  one  narrow  unstained  septum.     In  broth  cultures  it  often 
more  closely  resembles  B.  diphtherice,  but  under  all  other  conditions 
is  shorter.     It  forms  no  toxins  (Cobbett). 

2.  Hofmann's  bacillus  forms  no  acid  in  glucose  culture  media. 

3.  It  does  not  give  Neisser's  reaction  (see  pp.  476  and  481)  when 
grown  for  twenty-four  hours  on  Loffler's  ox  serum. 

4.  The  colonies  produced  by  Hofmann's  bacillus  on  blood  serum 
usually  become  after  a  few  days  larger,  more  opaque,  and  whiter 
than  those  of  the  diphtheria  bacillus.     They  also  grow  a  little  more 
rapidly  than  the  true  bacillus. 

5.  No  pathogenic  change  is  produced  in  guinea-pigs  inoculated 
with  this  bacillus  (1  c.c.  of  a  twenty-four  hours'  broth  culture),  and 
it  appears  to  be  innocuous  to  man.     In  forming  an  opinion,  all  the 
facts,  including  the  clinical,  if  possible,  must  be  taken  into  considera- 
tion.    But  on  the  whole,  recent  evidence  appears  to  support  the  view 
that  Hofmann's  bacillus  possesses  little,  if  any,  pathological  signifi- 
cance in  man.     It  does  not  agglutinate  like  B.  diphtherice. 

Attempts,  followed  by  some  degree  of  success,  have  been  made  to 
convert  a  virulent  diphtheria  bacillus  into  Hofmann's  bacillus,  and 
Hofmann's  bacillus  into  a  virulent  form  (Roux  ,and  Yersin,| 

*  For  a  fuller  statement,  see  Trans.  Jenner  Inst.  (First  Series),  pp.  7-32. 

f  For  a  discussion  of  the  three  forms  (a)  the  true  virulent  bacillus,  (b)  the  true 
non-virulent  bacillus,  and  (c)  the  pseudo-bacillus,  see  Report  of  Metropolitan  Asylums 
Board,  1901  (Gordon  Pugh). 

J  Ann.  de  Tlnst.  Past.,  1890,  vol.  iv. 


296  BACTERIA  AND  DISEASE 

Hewlett  and  Knight,*  Eichmond  and  Salter,f  Ohlmacher,J  and  others). 
Evidence  in  support  of  the  view  that  Hermann's  bacillus  is  an 
attenuated  variety  of  the  true  diphtheria  bacillus  has  been  brought 
forward.  But  it  can  only  be  accepted  provisionally.  Graham-Smith, 
Thomas  and  others  consider  the  pseudo-bacillus  to  be  absolutely 
innocuous.  In  practice,  it  is  the  right  course  at  present  to  look  upon 
the  presence  of  the  Hof mann  bacillus  as  indicating  a  suspicious  throat. 
It  should  not  be  forgotten  that  there  are  a  number  of  other  bacilli 
from  which  the  true  diphtheria  bacillus  must  be  differentiated.!  These 
include  the  B.  coryzce  segmentosus,  the  bacillus  of  Hofmann,  B.  xerosis, 
and  a  number  of  diphtheroid  bacilli,  and  organisms  from  nasal  and 
aural  discharge.  Similar  organisms  occur  in  birds  and  other  animals. 
There  are,  as  summarised  by  Gordon,  five  chief  characters  by  which 
the  true  diphtheria  bacillus  may  be  known: — (a)  The  macroscopic 
and  microscopic  appearance  of  the  growth  on  blood  serum;  (5)  the 
behaviour  of  the  bacillus  to  Loffler's  blue,  Gram's  stain,  and  Neisser's 
stain  for  granules ;  (c)  the  reaction  to  litmus  of  a  culture  in  alkaline 
broth,  containing  2  per  cent,  of  dextrose  after  48  hours  at  37°  C. ; 

(d)  the  pathogenic  test — 1  c.c.  of  broth  culture,  48  hours'  growth  at 
37°  C.,  injected  subcutaneously  into  200-300  gramme  guinea-pig,  pro- 
duces death  generally  in  48  hours,  whilst  post-mortem  heemorrhagic 
necrosis  and  oedema  are  found  locally,  the  internal  organs  are  con- 
gested, the  pleural,  pericardial,  and  peritoneal  fluids   are   increased, 
and  the  supra-renal  capsules  are  enlarged  and  engorged  with  blood ; 

(e)  the  virulence  of  the  organism  or  its  toxin  is  completely  neutralised 
by  a  simultaneous  dose  of  diphtheria  antitoxin.     For  purposes  of  rapid 
diagnosis,  (a)  and  (£)  are  relied  upon. 

Scarlet  Fever 

That  the  essential  cause  of  scarlet  fever  is  a  micro-organism  there 
can  be  little  doubt.  But  up  to  the  present  time  no  organism  has 
been  definitely  isolated  which  fulfils  the  postulates  of  Koch  in  respect 
to  specificity  of  bacteria.  Various  organisms  have,  however,  been 
described  as  associated  with  the  disease.  Edington,  Frankel,  Freud- 
enberg,  Klein,  Kurth,  Gordon,  Baginsky,  Class,  and  others  have 
described  organisms  which  they  believed  to  be  etiologically  related  to 
the  disease.  At  present,  however,  it  can  only  be  said  that  these 
bacteria  have  been  found  associated  with  scarlet  fever,  but  are  not  yet 
proved  to  be  its  cause.  The  organism  which  appears  at  present  to  be 

*  Trans.  .Tenner  Inst.  Prevent.  Med.  (First  Series),  1897,  p.  7  et  seq. 
f  Guy's  Hospital  Report,  1898,  vol.  liii.,  p.  55. 
j  Jour,  of  Med.  Research,  1902,  vol.  ii.,  p.  128. 

§  Rep.  of  Local  Govt.  Board,  1901-02,  pp.  418-39  (Gordon);  Jour,  of  /%.,  1904, 
pp.  299-316. 


SCARLET  FEVER  297 

the  most  likely  cause  of  the  disease  is  the  Streptococcus  scarlatinas  of 
Gordon.  Probably  the  organisms  isolated  by  Baginsky  and  Class  are 
different  forms  of  the  same  streptococcus. 

As  regards  dissemination,  it  has  long  been  known  that  scarlet 
fever,  like  small-pox,  is  most  commonly  spread  by  direct  infection 
through  the  medium  of  infected  clothing  and  other  articles,  or 
materials  handled  by  the  patient.  The  means  by  which  infection 
has  thus  been  carried  are  manifold,  and  need  not  claim  our  attention 
here.  As  we  have  seen,  in  1870  a  wider  field  of  conveyance  of  scarlet 
fever  was  revealed  by  the  investigations  of  Dr  M.  W.  Taylor  of 
Penrith.  While  studying  an  outbreak  of  scarlet  fever,  he  observed 
that  the  main  incidence  of  the  disease  fell  upon  customers  of  a  certain 
milk-shop  where  scarlet  fever  was  existent.  Since  that  date  abundant 
evidence  has  been  forthcoming  to  show  that  to  the  channels  of 
infection  previously  recognised,  that  of  conveyance  by  milk  must  be 
added.  Scarlet  fever  is  disseminated  in  many  ways  from  person  to 
person,  and  also  by  the  vehicle  of  "fomites."  The  virus  is  not 
diffusible,  but  is  evidently  tenacious  of  life.  Infected  garments  that 
have  been  put  aside  for  months  have  been  known  to  originate  an 
outbreak  of  the  disease.  Linen  has  been  known  on  many  occasions 
to  infect  laundresses.  There  is  no  evidence  that  the  virus  can  be 
conveyed  by  water.  As  a  rule,  probably  the  infection  of  scarlet  fever 
is  not  greatly  spread  by  aerial  connection,  but  by  articles  (toys,  books, 
bed-clothes,  letters,  etc.),  and  such  infected  articles  if  set  aside  in 
stagnant  air,  at  a  moderate  temperature,  and  in  the  absence  of  day- 
light, may  retain  the  infection,  like  garments,  for  months. 

Infectivity  begins  at  the  earliest  stage  of  the  attack,  but  is  prob- 
ably greatest  when  the  fever  is  at  its  highest.  In  most  cases  the 
patient  is  free  from  infection  at  the  end  of  six  weeks.  There  is  now 
strong  evidence  that  at  least  the  later  desquamation  is  not  infective. 
Probably  the  infection  lingers  longest  in  the  nasal,  tonsillar,  buccal, 
and  pharyngeal  mucus,  and  especially  in  any  chronic  discharge  from 
those  mucous  membranes.  Discharges  from  the  ear  may  retain 
infection  for  months.* 

It  is  most  probable  that  milk  obtains  its  infection  of  scarlet  fever 
from  being  brought  into  contact  with  persons  suffering,  as  a  rule, 
from  the  early  and  acute  stages  of  the  disease. 

Streptococcus  Scarlatinae  (Klein  and  Gordon).  Streptococcus  Conglomer- 
atus  (Kurth). — The  organism  was  isolated  from  the  blood,  nasal  and  tonsillar 
discharge  of  persons  suffering  from  scarlet  fever  in  its  earlier  and  later  stages.  Not 
from  urine  or  skin.  It  has  been  isolated  from  blood  of  persons  dying  from  scarlet 
fever.  Assumed  to  be  identical  with  streptococcus  isolated  from  diseased  udders  of 
cows  and  from  their  milk.  Found  by  Klein  in  ulcerations  of  teats  and  udders  of 

*  See  also  Report  to  Metropolitan  Asylums  Board  on  Return  Cases  of  Scarlet  Fever, 
by  W.  J.  Simpson,  M.D.,  1901,  p.  24  ;  also  Brit.  Med.  Jour.,  1902,  vol.  ii.,  p.  445 
(M.  H.  Gordon). 


298  BACTERIA  AND  DISEASE 

certain  cows.  Morphology. — A  streptococcus;  polymorphic;  showing  tendency  to 
oval  and  rod-shaped  elements,  especially  in  impression  preparations.  Presence  of 
wedge-shaped,  spindle-like,  rod-shaped  elements  in  agar  and  gelatine,  and  the 
characteristic  of  coherent  conglomeration  differentiate  this  streptococcus  from  others 
of  the  same  genus.  Irregularity  in  size  and  shape  of  elements  ;  every  transition 
between  coccus  and  bacillus.  Coccus  shape  prevails  in  bouillon,  the  bacillary  being 
more  common  on  agar.  The  streptococcus  is  stained  by  simple  stains  and  Gram's 
method.  Cultural  characters:  Bouillon. — At  37°  C.  after  24  hours,  the  medium 
remaining  clear,  a  single,  thick,  white-grey  mass,  or  several  smaller  masses,  appear 
at  the  foot  of  the  tube  ;  coherent  on  shaking  the  tube,  floats  through  the  medium  as 
a  flattened  bun-like  body.  Kurth  pointed  out  that  when  this  mass  was  examined 
under  the  microscope,  a  conglomerate  appearance  was  present.  The  mass  is  co- 
hesive. Gelatine  plates  and  tubes. — Slow  growth,  forming  small  grey  colonies, 
circular  or  oblong,  with  firm  edge,  and  consisting  of  closely  set  coherent  ^mass  of 
cocci.  Older  colonies  become  nodular.  Non -liquefy ing.  In  gelatine  at  37°  C.  the 
same  appearances  occur  as  in  bouillon,  but  often  more  marked.  Chain  formation  from 
these  cultures  is  more  marked  than  in  ordinary  streptococcus.  In  gelatine  at  37°  C.  this 
organism  grows  similar  to  S.  longus.  Agar  plates  and  tubes. — Three  types  of  colonies 
occur  after  24  hours :  (a)  grey,  granular,  irregularly-outlined  tuberculated  colonies  ; 
(ft)  colonies  of  similar  kind,  but  having  confluent  appearance  without  tubercles ; 
(c)  younger  and  smaller  colonies  which  have  a  fine  frilling  of  chains  around  a  more 
compact  coherent  centre.  The  most  useful  feature  for  differential  purposes  is  the 
granular,  glossy,  coherent  centre,  combined  with  tuberculation.  Grows  more  slowly 
than  8.  pyogenes^  and  on  the  whole  its  colonies  on  agar  are  smaller,  more  opaque,  and 
more  irregular  than  those  of  the  other  streptococci  present.  Milk. — Rapid  coagula- 
tion ;  produces  acid.  Sometimes  fails  to  clot  milk.  A  firm,  solid  clot  forms  Litmus 
milk,  as  a  rule,  within  the  first  2  days  at  37°  C.  After  24  hours  the  acid-production 
is  very  strong,  and  commonly,  when  there  is  a  clot  as  well,  the  lower  half  of  the  tube 
is  yellow-white— the  top  layer  being  pink.  This  decolorisation  of  lower  half  of  litmus 
milk  is  due  to  a  reducing  action  of  the  streptococcus.  Chain  formation  occurs  more 
than  in  bouillon.  The  four  chief  diagnostic  features  are  :  (1)  the  sediment  growth  in 
broth  cultures ;  (2)  the  rapid  coagulation  of  milk ;  (3)  the  acid  reaction  in  litmus 
milk;  (4)  the  character  of  the  agar  colonies.  Pathogenesis. — Pathogenic  for  mice 
and  rabbits.  After  passing  through  the  mouse,  the  streptococcus  takes  on  a  bacillary 
form  (Gordon),  and  other  modifications,  including  the  diminution  of  conglomeration, 
occur.  Its  virulence  differentiates  this  streptococcus  from  streptococci  present  in 
non-scarlatinal  throats,  except  S.  pyogenes ,  which  is  more  virulent  to  white  mice  than 
S.  conglomerate.  Klein  holds  that  this  S.  conglomerates  is  causally  related  to 
scarlet  fever  in  man,  and  is  wholly  distinct  from  8.  pyogenes.  Gordon  has  isolated 
the  latter  from  the  secretion  on  the  surface  of  the  tonsil  in  a  case  of  clinically  mild, 
uncomplicated  scarlatina.  It  has  also  been  found  like  the  S.  conglomeratus  in  the 
nasal  and  aural  discharge  of  scarlet  fever  patients.  Gordon  believes  that  both  strep- 
tococci may  play  a  part  in  the  causation  of  scarlet  fever,  but  that  S.  conglomeratus  is 
the  more  important  of  the  two,  and  that  it  occupies  a  position  in  the  bacteriological 
kingdom  between  S.  pyogenes  and  B.  diphtheria.  * 

Typhoid  Fever 

Typhoid  fever  is  an  acute  infectious  disease  characterised  clini- 
cally by  continuous  fever,  with  diarrhoea  and  other  symptoms,  and 
anatomically  by  a  more  or  less  extensive  ulceration  of  'the  Peyer's 
patches  in  the  intestine  (ileum),  with  swelling  of  the  mesenteric  glands 
and  enlargement  of  the  spleen.  The  lesion  of  importance  is  the  ulcera- 
tion of  the  bowel,  partly  on  account  of  its  origin,  partly  on  account  of 

*  For  full  record  of  Gordon's  researches,  see  Reports  of  Medical  Officer  to  Loc. 
Goo.  Bd.,  1898-99,  pp.  480-93  ;  1899-1900,  pp.  385-457  ;  and  1900-01,  pp.  353-404. 


TYPHOID  FEVER  299 

its  result.  The  pathogenetic  action  of  the  bacilli  is  obscure,  but  there 
can  be  no  doubt  that  the  ulcers  in  the  intestine  are  directly  or  in- 
directly the  result  of  the  specific  bacillus.  When  the  bacilli  reach  the 
intestine  they  multiply,  and,  penetrating  the  mucous  and  submucous 
coats,  set  up  the  changes,  which  lead,  first  to  hypersemia,  then  to  infil- 
tration, and  finally  to  ulceration  of  Peyer's  patches.  Some  of  the  bacilli 
pass  into  the  blood,  collecting  in  the  spleen  and  other  glands.  Whether 
in  the  bowels  or  in  the  organs  of  the  body,  the  bacilli  produce  their 
toxins,  and  as  a  result  of  their  action,  inflammation  and  fever  follow. 
The  inflammation  in  the  intestine  leads,  in  conjunction  with  the 
irritation  produced  by  the  ulcers,  to  increased  peristalsis,  and  there- 
fore diarrhoea.  Hence  the  excreta  of  a  typhoid  patient  have  two 
characteristics.  They  are  usually  abundant  and  frequent :  and  they 
are  charged  with  large  numbers  of  the  bacilli  of  typhoid  fever.  It 
is,  however,  necessary  to  guard  against  the  idea  that  typhoid  fever  is 
a  local  disease  of  the  intestine,  or  even  chiefly  so.  In  ordinary  cases, 
it  is  true,  the  intestinal  lesions  form  the  starting-point  of  the  disease, 
but  the  bacilli  rapidly  become  generalised,  and  are  found  in  the  most 
varied  parts  of  the  body,  and  not  uncommonly  in  the  blood  itself. 
Such  a  state  of  things  leads  to  a  condition  not  remote  from  septi- 
caemia, and  this  may  occur  with  little  or  no  local  lesion  in  the 
intestinal  tract.  The  reason  why  the  bacilli  of  typhoid  are  not  found 
in  greater  number  in  the  blood,  is  probably  in  part  due  to  the  fact 
that  in  ordinary  cases  the  blood  is  not  a  favourable  medium  for  their 
growth,  and  in  part  to  the  fact  that  they  are  rapidly  eliminated  or 
excreted.  "  Any  conception  of  the  disease,"  writes  Dr  Horton-Smith, 
"  which  regards  it  merely  as  affecting  the  alimentary  canal,  can  no 
longer  be  maintained.  On  the  contrary,  so  far  from  considering  it 
an  intestinal  disease,  pure  and  simple,  we  should  rather  look  upon  it 
as  a  modified  form  of  septicaemia.  It  is  septicaemia  in  that  always, 
and  in  all  cases,  the  bacilli  pass  into  the  blood,  and  then  into  the 
various  organs,  and  in  that  the  symptoms,  excepting  so  far  as  they 
are  intestinal,  are  referable  to  the  poisons  there  produced.  It  is  a 
modified  form,  however,  in  that  in  nearly  all  cases  there  is  a  definite 
local  and  primary  disease,  whence  the  secondary  dissemination  of 
the  micro-organism,  takes  place."* 

Whilst  it  has  been  held  that  typhoid  infection  can  pass  out  of 
the  infected  person  by  means  of  the  sweat,  the  expectoration,  the 
fasces  and  the  urine,  it  is  only  the  latter  two  which  need  be  considered 
as  a  rule.  Typhoid  stools  should  always  be  considered  infective, 
both  in  the  early  and  late  stages,  and  the  bacilli  have  even  been  found 
in  the  stools  fifteen  clays  after  the  temperature  has  become  normal. 
Further,  it  is  possible  that  the  typhoid  bacillus  may  be  distributed 

*  Brit.  Med.  Jour.,  1900,  i.,  pp.  827-34  (Gulstonian  Lectures,  1900),  P.  Horton- 
Smith. 


300  BACTERIA  AND  DISEASE 

by  persons  not  suffering  from  the  disease.  It  is  believed  that  the 
virus  of  typhoid  fever  is  chiefly  distributed  by  the  contents  of  the 
alimentary  canal,  and  this  view  is  so  universally  held  that  it  is 
unnecessary  to  elaborate  it. 

The  urine  is  the  other  chief  excretion  by  which  the  bacilli  of 
typhoid  fever  are  voided  from  the  body.  Horton-Smith  lias  demon- 
strated that  the  urine  of  typhoid  patients  contains  the  bacilli  of  the 
disease  in  the  proportion  of  one  in  every  four  cases.  He  has  also 
shown,  that,  as  a  rule,  it  is  towards  the  end  of  the  disease,  or  during 
convalescence,  that  this  condition  occurs.  Further,  whilst  it  is 
always  difficult  to  find  the  bacilli  in  the  stools,  in  the  urine  it  is 
generally  easy,  for  when  they  are  present  they  are  nearly  always  in 
pure  culture,  and  not  uncommonly  they  are  present  in  such  extra- 
ordinary numbers  that  one  cubic  centimetre  may  contain  many 
thousands  of  micro-organisms  (Horton-Smith).  Cammidge  found  37 
per  cent,  of  all  the  typhoid  urines  examined  contained  the  bacillus. 
In  one  case  the  organism  was  found  eight  months  after  convalescence. 
In  London,  Horton-Smith  found  typhoid  bacilli  present  in  the 
urine  in  25  per  cent,  of  all  cases  examined.  Working  in  Boston, 
Eichardson  obtained  a  positive  result  in  22*5  per  cent,  of  the 
cases  examined.  Both  the  last-named  investigators  found  the 
bacilli  present,  in  certain  cases,  in  such  large  numbers  that  the 
urine  was  rendered  turbid  by  their  presence.  Nor  are  such 
cases  rare.  Out  of  the  cases  in  which  the  specific  bacillus  was 
present  in  the  urine,  in  as  many  as  twelve  it  was  present  to  the 
degree  of  turbidity,  and  in  only  two  was  the  urine  described  as 
"  clear "  (Horton-Smith).  Referring  to  the  stage  of  the  disease  in 
which  the  bacilli  appear  in  the  urine,  they  have  been  found  as  early 
as  the  thirteenth  day  from  the  commencement,  and  as  late  as  the 
fourteenth  day  of  convalescence  (Horton-Smith).  Speaking  gener- 
ally, the  condition  is  rare  before  the  third  week  of  the  disease.  The 
duration  of  this  specific  bacilluria  also  varies.  The  shortest  duration 
recorded  by  Horton-Smith  was  eight  days,  but  in  four  other  cases  it 
had  not  disappeared  until  after  the  lapse  of  twenty-one  days,  twenty- 
five  days,  thirty  days,  and  seventy  days.  The  phenomenon  of  typhoid 
bacilli  in  the  urine  probably  occurs  because  one  or  more  bacilli  find 
their  way  into  the  bladder,  and  there  commence  rapid  growth  in  the 
urine  within  the  bladder,  which  medium  is  by  no  means  unfavour- 
able to  the  multiplication  of  the  bacillus  (Horton-Smith). 

From  these  facts  there  are  two  broad  deductions  which  concern 
the  bacteriologist  and  epidemiologist : — First,  that  enteric  fever 
occurs  as  a  result  of  infection  by  the  typhoid  bacillus  ;  and  secondly, 
that  the  typhoid  bacillus  leaves  the  body  of  the  infected  person 
through  two  chief  channels,  namely,  the  urinary  and  alimentary 
systems.  It  has  been  shown  further,  that  the  typhoid  bacillus  is 


TYPHOID  FEVER  301 

capable  of  a  saprophy tic  existence  in  soil,  dust,  water,  milk,  and  other 
natural  media.  It  can  survive  in  ordinary  earth  for  two  months, 
on  sterilised  linen  for  sixty  days,  on  woollen  cloth  for  eighty  days,  in 
sterilised  water  one  hundred  and  ninety-six  days,  in  particular  soils 
four  hundred  days  (Martin,  Firth  and  Horrocks,  etc.).  Therefore  it 
follows  that  the  organism  may  remain  in  the  body  for  long  periods, 
may  pass  from  the  body  in  urine  or  in  faeces,  and  find  its  way  into 
natural  media,  and  from  such  media,  sooner  or  later,  back  to  man. 
The  line  of  infection  may  be  direct  or  indirect ;  but  that  it  occurs 
there  can  be  no  doubt. 

The  Bacillus  of  Typhoid  Fever  (Eberth-Gaffky).— The  evidence 
that  Eberth's  bacillus  is  the  cause  of  typhoid  fever  consists  in  the 
main  of  three  parts : — (1)  The  bacillus  is  found  with  almost  invariable 
regularity  in  the  spleen  of  persons  dying  of  typhoid  fever,  when  an 
adequate  bacteriological  examination  is  made.  (2)  Eberth's  bacillus 
elaborates  specific  toxins.  These  toxins  are  for  the  most  part  intra- 
cellular,  contained  within  the  bacillus  itself,  and  are  chiefly  set  free 
when  the  latter  is  destroyed ;  and  they  are  comparatively  feeble 
compared  with  those  of  other  pathogenic  organisms ;  and  to  account 
for  the  clinical  conditions  of  the  disease  the  number  of  bacilli 
present  in  the  infected  body  would  have  to  be  exceptionally  large. 
This  fact,  coupled  with  the  varying  virulence  of  the  bacillus,  is  all 
the  more  remarkable  when  it  is  remembered  that  not  a  few  of  the 
epidemics  of  the  disease  have  arisen  from  a  dose  of  poison,  so 
excessively  minute  in  itself,  and  so  enormously  diluted,  as  to  appear 
out  of  all  proportion  to  the  number  of  persons  attacked.  It  is 
possible  that  a  few  organisms  introduced  into  the  human  body  are 
able,  under  certain  conditions,  to  multiply  rapidly,  and  so  bring 
about  the  same  results  as  large  dosage.  Again  and  again  it  has  been 
shown  that  considerable  epidemics  have  arisen  from  a  pollution  of 
water  so  slight  as  to  escape  detection  by  any  methods  of  chemical 
or  bacteriological  analysis  at  present  known.  (3)  The  blood  serum 
of  individuals  suffering  from  typhoid  fever  has  a  specific  agglutinative 
action  upon  the  Eberth  bacillus,  similar  to  that  observed  in  the 
blood  serum  of  animals,  rendered  immune  to  this  germ  (compare 
also  Pfeiffer's  reaction).  And  whilst  there  is  no  evidence  to  suppose 
that  animals  suffer  from  typhoid  fever  as  the  disease  occurs  in  man, 
there  is  evidence  to  show  that  under  certain  conditions,  a  disease, 
not  unlike  enteric  fever,  can  be  produced  by  inoculation  of  the  B. 
typhosus  into  guinea-pigs,  mice,  rabbits,  etc.  (Frankel  and  Simmonds). 
Klein  has  also  recently  demonstrated  by  inoculation,  that  the  bacillus 
is  able  to  multiply  and  develop  in  the  lymph-glands  of  the  calf. 
For  all  practical  purposes,  therefore,  the  B.  typhosus  of  Eberth  is 
now  generally  accepted  as  the  causal  agent  in  typhoid  fever. 

The  channels  of  infection  in  typhoid  fever  are  almost  entirely 


302  BACTERIA  AND  DISEASE 

j 

concerned  with  the  alimentary  tract.  Water,  milk,  shell-fish,  fried 
fish,  ice-cream,  watercress,  etc.,  have  all  been  proved  to  be  the 
vehicle  of  infection  in  spreading  the  disease.  Personal  contact  may 
and  does  operate  in  spreading  infection,  and  by  this  means  food 
also  may  become  contaminated.  Flies  are  held  to  have  acted  as 
carrying  agents  in  the  Spanish -American  War  of  1898  and  the 
Boer  War  of  1900-1902.  Oorfield  records  some  dozen  outbreaks  of 
typhoid  fever  due  to  general  insanitary  conditions,  60  outbreaks 
to  infected  water,  and  a  large  number  to  minor  channels.*  The 
writer  has  collected  160  records  of  milk-borne  outbreaks  of  the 
disease.f 

In  1880-81  Eberth  announced  the  discovery  of  the  typhoid 
bacillus  in  cases  of  clinical  enteric  fever.  In  1884  it  was  first 
cultivated  outside  the  body  by  G-affky.  Since  then  other  organisms 
have  been  held  responsible  for  the  causation  of  enteric  (or  typhoid) 
fever.  In  1885  the  B.  coli  communis  was  recognised,  and  it  has 
been  a  matter  of  some  debate  among  bacteriologists  as  to  how  far 
these  two  organisms  are  the  same  species,  and  interchangeable. 
There  is  evidence  on  both  sides  of  the  question,  but  bacteriologists 
generally  regard  the  Eberth-Gaffky  bacillus  as  the  specific  cause  of 
typhoid  fever,  though  complete  proof  is  still  wanting. 

Under  the  microscope  the  bacilli  appear  as  rods,  2-4  yu  long, 
•5  /x  broad,  having  round  ends.  Sometimes  threads  are  observable, 
being  10  /x  in  length.  In  the  field  of  the  microscope  the  bacilli 
differ  in  length  from  each  other,  but  are  approximately  of  the  same 
thickness.  Bound  and  oval  cells  constantly  occur  even  in  pure 
culture,  and  many  of  these  shorter  forms  of  typhoid  appear  to  be 
identical  in  morphology  with  some  of  the  many  forms  of  B.  coli. 
There  are  no  .spores.  Motility  is  marked ;  indeed,  in  young  culture 
the  typhoid  bacillus  is  the  most  active  pathogenic  germ  we  know. 
The  small  forms  move  about  with  extreme  rapidity;  the  longer 
forms  move  in  a  vermicular  manner.  Its  powers  of  motility  are 
due  to  some  five  to  twenty  flagella  of  varying  length,  some  of  them 
being  much  longer  than  the  bacillus  itself.  The  flagella  are  both 
terminal  and  lateral,  and  are  elastic  and  wavy. 

The  organism  may  be  isolated  from  the  ulcerated  Peyer's  patches 
in  the  intestine,  from  the  spleen,  the  mesenteric  glands,  and  the 
urine.  Owing  to  the  mixture  of  bacteria  found  elsewhere,  it  is 
generally  most  readily  isolated  from  the  spleen.  The  whole  spleen 
is  removed,  and  a  portion  of  its  capsule  seared  with  a  hot  iron  to 
destroy  superficial  organisms.  With  a  sterilised  knife  a  small  cut 
is  made  into  the  substance  of  the  organ,  and  by  means  of  a  sterilised 
platinum  wire  a  little  of  the  pulp  is  removed  and  traced  over  the 

*  The  Milroy  Lectures  on  Typhoid  Fever,  1902. 

j  Bacteriology  of  Milk,  1903  (Swithinbank  and  Newman). 


PLATE  20. 


*.-£$:?**•  b 

t**3&-$j"x     $X      ~ 

^*rf?.33&£ .  & 


Vfcw  *fiJK™*<»?2 
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typhosus.  Film  preparation  from  agar,  16 
hours  at  37°  C.  Stained  with  carbol  fuchsin 
X  1000. 


" 


>AL-GRUBER  REACTION.     Agglutination  of  B. 

typhosus    by    blood    serum    of    a    typhoid  Bacillus 

patient,     x   400. 


Bacillus  typhosus,  showing  flagella.     x  1000. 


is.    From  human  mesenteric  gland.     Stained  with 
methylene  blue,     x  750. 


(To  face  page  302, 


TYPHOID  FEVER  303 

surface  of  agar.  The  agar  reveals  a  growth  in  about  twenty-four 
hours  at  37°  C.,  which  is  the  favourable  temperature.  A  greyish, 
moist,  irregular  growth  appears,  but  it  is  invariably  attached  to 
the  track  of  the  inoculating  needle.  On  agar  plates  the  superficial 
colonies  appear  as  circular  spots,  dull  white  by  reflected  light  and 
bluish-grey  by  transmitted  light.  The  deep  colonies  are  opaque 
and  finely  granular.  On  gelatine  the  growth  is  much  the  same,  but 
its  irregular  edge  is,  if  anything,  more  apparent.  There  is  no 
liquefaction  and  no  gas  formation.  On  plates  of  gelatine  the 
colonies  become  large  and  spreading,  with  wavy  margins.  The  whole 
colony  appears  raised  and  almost  limpet-shaped,  with  delicate  lines 
passing  over  its  surface.  When  magnified,  there  is  an  appearance 
of  transparent  iridescence.  The  growth  on  potato  is  termed 
"  invisible,"  and  is  of  the  nature  of  a  potato-coloured  pellicle,  which 
appears  to  be  moist,  and  may  at  a  later  stage  become  light  brown 
in  colour,  particularly  if  the  potato  is  fresh.  Milk  is  a  favourable 
medium,  and  is  rendered  slightly  acid.  No  coagulation  takes  place. 
Broth  is  rendered  turbid,  and  there  is  the  formation  of  a  sediment. 
The  organism  is  stained  with  carbol-thionin  blue,  carbolic  fuchsin, 
etc.  It  is  decolorised  by  Gram's  methods. 

The  problem  of  isolating  the  typhoid  bacillus  is  greatly  com- 
plicated by  the  fact  that  B.  coli  communis  is  a  normal  inhabitant 
of  the  alimentary  canal,  is  widely  distributed  in  nature,  and  is  in 
many  respects  similar  to  the  typhoid  bacillus.  (For  full  account 
of  B.  coli  and  its  similarities  to  the  typhoid  bacillus,  see  pp.  46-51.) 

We  have  pointed  out  elsewhere  the  relation  between  soil  and 
typhoid.  In  water,  even  though  we  know  it  is  a  vehicle  of  the 
disease,  the  Bacillus  typhosus  has  been  only  very  rarely  detected. 
The  difficulties  in  separating  the  bacillus  from  water  (like  that  at 
Maidstone,  for  example),  which  appears  definitely  to  have  been  the 
vehicle  of  the  disease,  are  manifold.  To  begin  with,  the  enormous 
dilution  must  be  borne  in  mind,  a  comparatively  small  amount  of 
contamination  being  introduced  into  large  quantities  of  water. 
Secondly,  the  group  of  the  B.  coli  species  considerably  complicates 
the  search. 

Further,  we  must  bear  in  mind  a  point  that  is  frequently  neglected, 
namely,  that  the  bacteriological  examination  of  a  water  which  is 
suspected  of  having  conveyed  the  disease  is  from  a  variety  of  circum- 
stances conducted  too  late  to  detect  the  causal  bacteria.  The  in- 
cubation period  of  typhoid  we  may  take  at  fourteen  days.  Let  us 
suppose  a  town  water  supply  is  polluted  with  some  typhoid  excreta 
on  the  1st  of  January.  Until  the  14th  of  January  there  may  be 
no  knowledge  whatever  of  the  state  of  affairs.  Two  or  three  days 
are  required  for  notification  of  cases.  Several  more  days  elapse 
generally  before  bacteriological  evidence  is  demanded.  Hence  arises 


304  BACTERIA  AND  DISEASE 

the  anomalous  position  of  the  bacteriologist  who  sets  to  work  to 
examine  a  water  suspected  of  typhoid  pollution  three  weeks 
previously.  There  can  be  no  doubt  that  these  three  difficulties  are 
very  real  ones.  The  solution  to  the  problem  will  be  found  in  the 
dictum  that  "  a  water  in  which  sewage  organisms  have  been  detected 
in  large  numbers  should  be  regarded  with  suspicion  "  as  the  vehicle  of 
typhoid,  even  though  no  typhoid  bacilli  are  discoverable.  The  chief 
of  these  sewage  bacteria  are  believed  to  be  Proteus  vulgaris,  Bacillus 
coli,  Proteus  Zcnkcri,  and  Bacillus  enteritidis  sporogenes. 

It  may  occur  to  the  reader  that,  as  the  typhoid  bacillus  is,  as  far 
as  we  know,  comparatively  common,  drinking  water  may  frequently 
act  as  a  vehicle  to  carry  the  disease  to  man.  But,  to  appreciate  the 
position,  it  is  desirable  to  bear  in  mind  the  following  facts.  The 
typhoid  bacillus  is  found,  with  other  bacteria,  in  the  excrement  of 
patients  suffering  from  the  disease ;  it  is  short-lived ;  in  waters  there 
exist  organisms  which  can  exert  an  influence  in  diminishing  its 
vitality ;  it  is,  so  to  speak,  enormously  diluted  in  waters ;  exposure 
to  direct  sunlight  destroys  it;  and  it  has  a  tendency  to  be  carried 
down  stream,  or  in  still  waters  settle  to  the  bottom  by  subsidence. 
Even  when  all  the  conditions  are  fulfilled,  it  must  not  be  forgotten 
that  a  certain  definite  dose  of  the  bacillus  is  required  to  be  taken, 
and  that  by  a  "  susceptible  "  person. 

Epidemic  Diarrhoea 

By  "epidemic  diarrhoea"  (zymotic  or  epidemic  enteritis)  is  meant 
a  specific  disease,  which  may  be  defined  as  an  acute  infective 
disease,  affecting  chiefly  children  Under  two  years  of  age,  occurring 
during  the  summer  months  in  epidemic  form,  and  characterised  by 
the  occurrence  of  diarrhoea,  vomiting,  and  wasting,  accompanied  in 
severe  cases  by  toxsemia  and  collapse.  The  disease  is  a  large 
contributor  to  infant  mortality,  and  in  many  urban  districts  it  is 
the  most  serious  of  all  infant  diseases,  if  measured  by  fatality. 

The  exact  cause  of  epidemic  diarrhoea  is  not  at  present  known. 
In  1887  Ballard  formulated  certain  propositions  which  have 
obtained  general  acceptance.  They  are  as  follow : — 

"  That  the  essential  cause  of  diarrhoea  resides  ordinarily  in  the 
superficial  layers  of  the  earth,  where  it  is  intimately  associated  in 
the  life-processes  of  some  micro-organism  not  yet  detected  or 
isolated. 

"  That  the  vital  manifestations  of  such  organism  are  dependent, 
among  other  things,  perhaps  principally  upon  conditions  of  season 
and  on  the  presence  of  dead  organic  matter,  which  is  its  pabulum. 

"That  on  occasion  such  micro-organism  is  capable  of  getting 
abroad  from  its  primary  habitat,  the  earth,  and  having  become  air- 


EPIDEMIC  DIARRHCEA  305 

borne,  obtained  an  opportunity  for  fastening  on  non-living  organic 
material,  and  of  using  such  organic  material  both  as  nidus  and  as 
pabulum  in  undergoing  various  phases  of  its  life-history. 

"  That  in  food,  inside  as  well  as  outside  the  human  body,  such 
micro-organism  finds,  especially  at  certain  seasons,  nidus  and 
pabulum  convenient  for  its  development,  multiplication,  or  evolu- 
tion. 

"  That  from  food,  as  also  from  the  contained  organic  matter  of 
particular  soils,  such  micro-organism  can  manufacture  by  the 
chemical  changes  wrought  therein  through  certain  of  its  life- 
processes  a  substance  which  is  a  virulent  chemical  poison;  and 
that  this  chemical  poison  is,  in  the  human  body,  the  material 
cause  of  epidemic  diarrhoea."* 

Bacteriology  of  Diarrhoaa. — The  three  causal  agents  which 
Ballard  mentions  as  playing  a  large  part  in  the  production  of  this 
disease  are  the  soil,  season,  and  food — and  the  causa  causans  is 
"some  micro-organism  not  yet  detected  or  isolated."  It  must  be 
said  that  we  have  not  got  much  further  than  this  during  the  last 
fifteen  years. 

In  1885  Escherich  published  his  classical  researches  on  B.  coli 
communis.  He  pointed  out  that  the  meconium  of  the  newly-born 
infant  is  free  from  bacteria,  but  by  the  second  day  they  are  present 
in  large  numbers,  and  in  the  ordinary  excreta  of  healthy  infants  he 
found  chiefly  two  organisms,  B.  lactis  cerogenes  and  B.  coli  communis. 
Of  these  the  former  was  the  more  abundant  in  the  upper  part  of 
the  small  intestine,  and  the  latter  in  the  lower  part  and  in  the 
colon,  so  that  in  the  excreta  B.  coli  was  abundant,  and  B.  lactis 
comparatively  scarce.  Booker,  working  in  1886  and  onwards,  found 
that  the  constant  bacteria  of  the  healthy  excreta  of  the  infant 
(B.  coli  and  B.  lactis  cerogenes)  do  not  disappear  in  the  excreta  of 
diarrhoea.  B.  coli,  however,  does  not  predominate  in  the  same 
degree,  and  B.  lactis  is  present  generally  in  greater  numbers  than 
in  the  healthy  excreta.  Booker  examined  the  excreta  of  31 
children,  and  isolated  33  different  species  of  bacteria.  Many 
varieties  of  bacteria  are  sometimes  found  in  individual  cases  of 
diarrhoea.  The  greatest  number  were  found  in  cases  of  cholera 
infantum,  and  a  larger  number  in  catarrhal  enteritis  than  in 
dysenteric  enteritis.  The  actual  number  of  individual  bacteria 
was,  he  found,  as  great  in  the  healthy  excreta  as  in  the  diarrhceal 
excreta.  Proteus  vulgaris  was  found  very  generally,  and  in  the 
most  serious  cases.  No  chromogenic  bacteria  were  isolated,  and 
cultures  from  a  large  number  of  green  stools  failed  to  develop  green 
colonies.  From  these  facts  Booker  concluded  "  that  not  one  specific 

*  Supplement  to  the  Report  of  the  Medical  Officer  of  the  Local  Government  Board. 

1887. 

U 


306  BACTERIA  AND  DISEASE 

organism,  but  many  different  varieties  of  bacteria,  are  concerned  in 
the  etiology  of  the  summer  diarrhoeas  of  children. 

From  1889-1895  Booker  continued  his  studies,  isolating  bacteria 
from  the  rectum  in  92  infants  affected  with  epidemic  diarrhoea,  and 
also  from  the  organs  of  33  infants  who  died  from  this  disease.  He 
found  the  conditions  for  the  development  of  bacteria  in  the 
intestine  of  infants  affected  with  summer  diarrhoea  different  from 
those  in  the  healthy  intestine  of  milk-fed  infants,  in  that  they 
favoured  more  varied  bacterial  vegetation,  a  rich  growth  of  the 
inconstant  species  of  intestinal  bacteria,  and  a  more  uniform  dis- 
tribution through  the  intestine  of  the  two  constant  varieties  of 
healthy  excreta  bacteria  {B.  coli  communis  and  B.  lactis  cerogenes). 
The  first  step  in  the  pathological  process,  Booker  believes  to  be  a 
direct  injury  to  the  epithelium  from  abnormal  or  excessive  fermenta- 
tion and  from  toxic  products  of  bacteria ;  and  secondly,  a  general 
intoxication  may  be  brought  about  indirectly  through  the  production 
of  soluble  poisons.  He  holds  that,  bacteriologically  and  anatomically, 
three  principal  forms  of  summer  diarrhoea  of  infants  may  be 
provisionally  distinguished: — (i.)  dyspeptic  or  non-inflammatory 
diarrhoea ;  (ii.)  streptococcal  gastro-enteritis ;  and  (iii.)  bacillary 
gastro-enteritis.*  As  a  result  of  his  extended  researches,  Booker 
came  to  a  general  conclusion  which  he  expressed  as  follows : — "  No 
single  micro-organism  is  found  to  be  the  specific  excitor  of  the 
summer  diarrhoea  of  infants,  but  the  affection  is  generally  to  be 
attributed  to  the  activity  of  a  number  of  varieties  of  bacteria,  some 
of  which  belong  to  well-known  species,  and  are  of  ordinary  occurrence 
and  wide  distribution,  the  most  important  being  the  streptococcus 
(enteritidis)  and  Proteus  vulgaris."  The  streptococcus  termed  S. 
enteritidis  varies  in  morphology,  and  seems  to  be  associated  with 
two  classes  of  cases,  one  of'  which  simulates  cholera,  the  other 
typical  enteric  fever.  "Micrococci  are  present  in  all  cases,  some- 
times in  enormous  numbers."  •]•  It  may  be  added  that  Cumston, 
Hoist,  Escherich,  and  Hirsch  have  also  laid  emphasis  upon  the 
causal  relationship  of  certain  streptococci  and  diarrhoea. 

Klein  was  one  of  the  first  workers  to  isolate  an  anaerobic 
organism  from  cases  of  epidemic  diarrhoea.  This  organism,  which 
he  named  B.  enteritidis  sporogenes,  was  found  in  three  successive 
outbreaks  of  diarrhoea  occurring  among  patients  in  St  Bartholomew's 
Hospital.  In  the  first  two  outbreaks  the  milk  was  evidently  the 
channel  of  infection,  in  the  third  it  was  some  rice  pudding.  The 

*  Johns  Hopkins  Hospital  Reports,  1896,  vol.  vi.,  p.  253.  See  also  a  paper  "  On 
the  Growth  of  Bacteria  in  the  Intestine,"  by  Lorrain  Smith  and  Tennant — Brit. 
Med.  Jour.,  1902,  vol.  ii.,  p.  1941.  Also  Jeffries,  Trans.  American  Pediatrics 
Society,  vol.  i.,  1889;  Baginsky,  Archiv.  f.  Kinderheilkunde,  xii.,  Nos.  1  and  2; 
Berliner  klin.  Woch.,  1889  ;  and  Flexner  and  Holt's  Rockefeller  Inst.  Rep.,  1904. 

f  Johns  Hopkins  Hospital  Reports,  1896,  vol.  vi.,  p.  251. 


PLATE  21, 


&          0 


; 


/ 


teritidia  sporogenes.    (Klein.) 
Film  preparation  from  serum  culture,  showing  spores.     X  2000. 


ANAEROBIC  MILK  CULTURE,  SHOWING  THE  "  ENTERITIDIS  CHANCE.'     (Klein.) 
From  left  to  right,  tubes  contain  ,>,,  ,}„,  ,J,,,  „,}„„  c.c.  ot  Nottingham  crude  sewage. 

[To  face  page  307. 


EPIDEMIC  DIARRHCEA  307 

bacillus  was  carefully  studied,  and  the  main  facts  respecting  it  may- 
be stated  briefly  here : — 

B.  enteritidis  sporogenes  (Klein)  is  an  anaerobic  bacillus:  1'6-4'S  /a  long,  and 
0'8  Abroad;  stains  by  Gram's  method  and  ordinary  stains.  Motile;  spore  forma- 
tion present ;  large,  oval  spores  often  situated  near  one  end  of  bacillus  ;  grows  well 
on  gelatine  and  agar.  In  the  former  gas  is  produced  and  the  gelatine  liquefies. 
On  agar  there  is  also  gas  fermentation,  and  the  colonies  in  the  depth  are  round, 
white  by  reflected  light,  brown  and  granular  in  transmitted  light.  On  the  surface 
of  agar  plates  flat,  circular,  moist,  grey  colonies  appear  in  twenty-four  to  forty- 
eight  hours ;  thicker  in  centre  than  at  margin,  and  showing  granularity.  Bacillus 
grows  well  in  milk,  producing  the  "enteritidis  change."  After  thirty-six  hours  of 
anaerobic  incubation  at  37°  C.  in  milk,  the  cream  is  torn  or  disassociated  by  develop- 
ment of  gas,  so  that  the  surface  of  the  medium  is  covered  with  stringy,  pinkish- 
white  masses  of  coagulated  casein  enclosing  a  number  of  gas-bubbles.  The  main 
portion  of  the  tube  of  milk  contains  a  colourless,  thin,  watery  whey,  with  a  few 
casein  lumps  here  and  there  adhering  to  the  sides  of  the  tube.  The  whey  has  a 
smell  of  butyric  acid,  and  is  acid  in  reaction.  It  contains  many  bacilli.  Patho- 
genesis — If  1  c.c.  of  milk  whey  containing  the  bacillus  be  injected  into  a  guinea-pig 
(200  to  300  grammes),  a  swelling  appears  in  six  hours,  extending  over  abdomen 
and  thigh,  and  death  occurs  in  eighteen  to  twenty-four  hours.  Post  mortem  :  sub- 
cutaneous gangrene,  with  much  sanguineous  exudation,  in  which  bacilli  and  spores 
will  be  found.  Klein  considers  this  organism  to  be  the  cause  of  epidemic  diarrhoea. 

B.  enteritidis  sporogenes  is  a  widely-distributed  organism,  and 
occurs  in  normal  and  typhoid  excreta,  in  sewage,  manure,  soil,  dust, 
and  milk.*  The  etiological  relationship  between  this  bacillus  and 
epidemic  diarrhoea  has  been  called  in  question,  and  it  is,  of  course, 
not  proved  that  the  organism  is  the  cause  of  the  disease.  On  the 
other  hand,  it  has  been  very  frequently  found  in  the  mucous  flakes 
of  the  dejecta  in  patients  suffering  from  the  disease,  and  in  the 
outbreak  produced  by  the  consumption  of  cooked  rice  pudding  it 
is  difficult  to  understand  how  any  organism  except  an  anaerobe  of 
highly  resistant  qualities  could  have  produced  the  condition.  It 
will  be  apparent,  moreover,  that  B.  enteritidis  sporogenes  fulfils  in  a 
somewhat  exceptional  degree  the  requirements  suggested  by  Ballard. 

That  epidemic  diarrhoea  is  caused  by  the  B.  coli  either  alone  or 
in  conjunction  with  other  organisms,  has  been  held  by  a  number  of 
authorities.  Gums  ton,  who  investigated  13  cases  of  the  disease,  con- 
cluded that  B.  coli  associated  with  Streptococcus  pyogenes  was  the 
chief  pathogenic  agent  concerned,  and  he  claims  that  the  virulence  of 
B.  coli  is  exalted  by  the  association. f  Lesage  also  formed  the  opinion 
that  the  disease  was  due  to  B.  coli,  and  investigated  the  agglutinative 
properties  of  the  serum  of  children  suffering  from  epidemic  diarrhoea 
on  B.  coli  isolated  from  the  intestine.  He  obtained  a  positive  result 
in  40  out  of  50  cases,  and  the  serum  of  each  of  these  40  cases,  also 
agglutinated  samples  of  B.  coli  from  39  other  children  seized  with 

*  Reports  of  Medical  Officer  of  Local  Government  Board,  1897-98,  pp.  210-51 ; 
1902,  p/406. 

t  International  Medical  Magazine,  February  1897. 


308  BACTERIA  AND  DISEASE 

the  same  disease.*  Some  of  the  most  recent  work  on  the 
relationship  existing  between  B.  coli  and  epidemic  diarrhoea  has 
been  done  by  Delepine,  who  examined  milk  in  the  outbreak  of 
epidemic  diarrhoea  which  occurred  in  Manchester  in  1894  (see 
p.  224),  and  has  also  examined  a  large  number  of  town  and  country 
milks.  His  conclusion  is  that : — 

"  Epidemic  diarrhoea  of  the  common  type  occurring  in  this  country 
is  apparently,  in  the  great  majority  of  instances,  the  result  of 
infection  of  food  by  bacilli  belonging  to  the  colon  group  of  bacilli, 
and  which  are  present  at  times  in  foecal  matter.  It  appears  that 
this  infection  of  food  does  not  generally  lead  to  serious  consequences, 
unless  the  infection  is  massive  from  the  first,  or  the  food  is  kept 
for  a  sufficient  length  of  time,  and  under  conditions  of  temperature 
favouring  the  multiplication  of  these  bacilli. 

"Milk,  which  is  the  most  common  cause  of  epidemic  diarrhoea 
in  infants,  is  usually  infected  at  the  farm,  or  (through  vessels)  in 
transit.  Of  the  bacilli  of  the  colon  group  which  are  capable  of 
rendering  the  milk  infectious,  those  which  do  not  produce  a  large 
amount  of  acid,  and  do  not  coagulate  milk,  are  the  most  virulent, 
and  are  probably  the  essential  cause  of  epidemic  diarrhoea."  f 

It  is  evident  that  our  knowledge  of  the  bacteriology  of  diarrhoea 
is  not  sufficiently  established  to  permit  of  any  very  definite  con- 
clusion on  the  matter.  It  may  be  that  the  whole  group  of  choleraic, 
enteric,  and  diarrhoeal  diseases  are  caused  by  a  group  of  micro- 
organisms having  many  similarities  and  relationships  to  each  other ; 
or  it  may  be  that  different  forms  of  diarrhoea  have  their  own  specific 
causal  organism;  or,  lastly,  it  may  be  a  question  of  association  of 
organisms  or  of  toxins  which  brings  about  the  disease.^  In  any 
event,  there  is  abundant  evidence  that  epidemic  diarrhoea  is  a 
bacterial  disease  in  the  same  sense  as  typhoid  fever. 

Conditions  favourable  to  Epidemic  Diarrhoea. — The  pro  visional 
results  of  Ballard's  inquiry  into  the  causation  of  epidemic  diarrhoea 
may  be  stated  as  follows : — 

"  The  summer  rise  of  diarrhoeal  mortality  does  not  commence 
until  the  mean  temperature  recorded  by  the  4-foot  earth  thermometer 
has  attained  somewhere  about  56°  F.,  no  matter  what  may  have  been 
the  temperature  previously  attained  by  the  atmosphere  or  recorded 
by  the  1-foot  earth  thermometer.  The  maximum  diarrhoeal  mortality 
of  the  year  is  usually  observed  in*  the  week  in  which  the  temperature 
recorded  by  the  4-foot  earth  thermometer  attains  its  mean  weekly 
maximum.  The  decline  of  the  diarrhoeal  mortality  is  in  this  con- 

*  La  Semaine  Med.,  October  1897. 
t  Jour,  of  Hygiene,  1903,  vol.  iii.,  No.  1,  p.  90. 

t  See  also  Report  of  Medical  Officer  to  Local  Government  Board,  1902,  p.  395 
(Martin),  404  et  sey.  (Klein). 


EPIDEMIC  DIARRHCEA  309 

neotion  not  less  instructive,  perhaps  more  so,  than  its  rise.  It 
coincides  with  the  decline  of  the  temperature  recorded  by  the  4-foot 
earth  thermometer,  which  temperature  declines  very  much  more 
slowly  than  the  atmospheric  temperature,  or  than  that  recorded  by 
the  1-foot  thermometer;  so  that  the  epidemic  mortality  may  con- 
tinue (although  declining)  long  after  the  last-mentioned  temperatures 
have  fallen  greatly,  and  may  extend  some  way  into  the  fourth 
quarter  of  the  year.  The  atmospheric  temperature  and  the  tempera- 
ture of  the  more  superficial  layers  of  the  earth,  is  little  if  at  all 
apparent  until  the  temperature  recorded  by  the  4-foot  earth  ther- 
mometer has  risen  as  stated  above ;  then  their  influence  is  apparent, 
but  it  is  a  subsidiary  one." 

In  addition  to  these  conditions  of  soil,  Ballard  and  other  workers 
have  concluded  that  insanitation  in  the  widest  sense  of  the  term 
favours  epidemic  diarrhoea.  Density  of  population  or  houses  upon 
an  area,  unclean  soil,  dusty  surfaces,  bad  light,  absence  of  ventilation, 
maternal  neglect,  etc.,  all  have  a  share  in  creating  an  environment 
favourable  to  the  disease.  As  we  have  seen,  Delepine,  like  Ballard, 
attributes  the  disease  in  large  measure  to  milk.  Ballard  believed 
that  milk  gained  its  infection  by  unsuitable  storage  and  by  the  mode 
in  which  it  was  used.  He  found  that  "  infants  fed  solely  from  the 
breast  are  remarkably  exempt  from  fatal  diarrhoea ;  that  infants  fed 
in  whatever  way  with  artificial  food  to  the  exclusion  of  breast  milk 
are  those  which  suffer  most  heavily  from  fatal  diarrhoea;  that 
children  fed  partially  at  the  breast  and  partially  with  other  kinds 
of  food,  suffer  to  a  considerable  extent  from  fatal  diarrhoea,  but  very 
much  less  than  those  brought  up  altogether  by  hand ;  and  that,  as 
respects  the  use  of  '  the  bottle/  it  is  decidedly  more  dangerous  than 
artificial  feeding  without  the  use  of  the  bottle."  This  view  has  been 
confirmed  by  Newsholine,  Mven,  Eichards,  the  writer,  and  others. 

Dr  Newsholme  of  Brighton  has  published  an  interesting  paper 
on  the  causation  of  epidemic  diarrhoea.  Some  of  his  chief  conclusions, 
which  are  now  widely  accepted,  may  be  added : — 

"(1)  Epidemic  diarrhoea  is  chiefly  a  disease  of  urban  life.  (2) 
Epidemic  diarrhoea  as  a  fatal  disease,  is  a  disease  of  the  artisan  and 
still  more  of  the  lower  labouring  classes  to  a  preponderant  extent. 
This  is  probably  largely  a  question  of  social  status  per  se ;  that  is, 
it  is  due  to  neglect  of  infants,  uncleanly  storage  of  food,  industrial 
occupation  of  mothers,  etc.  (3)  Towns  which  have  adopted  the 
water-carriage  system  of  sewerage  have,  as  a  rule,  much  less  diarrhoea 
than  those  retaining  other  methods  of  removal  of  excrement.  (4) 
Towns  with  the  most  perfect  scavenging  arrangements,  including  the 
methods  of  removal  of  house  refuse,  have  the  least  epidemic  diarrhoea. 
It  has  recently  been  suggested  that  epidemic  diarrhoea  is  due  to 
surface  pollution  derived  from  street  dust,  particularly  dried  horse- 


310  BACTERIA  AND  DISEASE 

manure  (Waldo).  (5)  The  influence  of  the  soil  is  a  decided  one. 
Where  the  dwelling-houses  of  a  place  have  as  their  foundation  solid 
rock,  with  little  or  no  superincumbent  loose  material,  the  diarrhoeal 
mortality  is,  notwithstanding  many  other  unfavourable  conditions 
and  surroundings,  low.  On  the  other  hand,  a  loose  soil  is  a  soil  on 
which  diarrhoeal  mortality  is  apt  to  be  high  (Ballard).  The  pollution 
of  soil  is  probably  the  important  element  in  the  causation  of  diarrhoea 
in  towns  on  pervious  soils.  (6)  Given  two  towns  equally  placed  so 
far  as  social  and  sanitary  conditions  are  concerned,  their  relative 
diarrhoeal  mortality  is  proportional  to  the  height  of  the  temperature 
and  the  deficiency  of  the  rainfall  in  each  town,  particularly  of  the 
third  quarter  of  the  year." 

Dr  Newsholme  concludes  that  "  the  fundamental  condition  favour- 
ing epidemic  diarrhoea  is  an  unclean  soil,  the  particulate  poison  from 
which  infests  the  air  and  is  swallowed,  most  commonly  with  food, 
especially  milk."  In  other  words,  epidemic  diarrhoea  is  a  so-called 
"  tilth-disease,"  preventable  by  improved  sanitation  in  the  broadest 
meaning  of  the  term.* 

From  the  facts  and  suggestions  quoted  above,  and  they  are  but 
representative  of  many  other  similar  views  receiving  the  general 
support  of  epidemiologists,  it  will  be  evident  that  at  the  present  time 
the  cause  of  epidemic  diarrhoea  is  to  be  found  in  four  conditions, 
which  may  be  expressed  shortly  as  two  propositions,  thus :  (1) 
Epidemic  diarrhoea  is  a  bacterial  disease ;  (2)  its  occurrence  depends, 
wholly  or  partly,  upon  surrounding  temperature,  deficiency  of  rain- 
fall, and  pollution  of  food,  chiefly  milk.  The  exact  relationship 
which  these  conditions  have  to  each  other  is  not  known.  Some 
authorities  hold  that  a  certain  temperature  affects  food,  conducing 
towards  creating  in  it  injurious  properties.  Others  believe  that  it 
is  a  question  of  pollution  of  milk  by  dust,  which  carries  to  the  milk 
the  causal  micro-organisms,  and  that  deficient  rainfall  favours  this 
contamination,  and  increased  temperature  favours  the  growth  and 
multiplication  of  the  bacteria  thus  conveyed  to  the  milk.  As  Dr 
Newsholme  says,  "Whatever  be  its  mode  of  operation,  a  frequent 
fall  of  rain  during  the  summer  weeks,  even  though  its  total  amount 
be  not  great,  is  one  of  the  most  effectual  means  of  keeping  down 
the  diarrhoeal  death-rate  ";  f  and  whilst  he  considers  temperature 
conditions  of  great  importance,  "rainfall  is  more  important  than 
temperature  in  relation  to  epidemic  diarrhoea."  Kain  washes  the  air, 
if  the  expression  may  be  allowed,  and  carries  to  the  surface  aerial 
dust.  It,  of  course,  also  washes  the  surface  of  the  soil  and  removes 
surface  pollution,  and  with  it  micro-organisms  capable  of  infecting 
infants,  usually  by  food.  Thus  the  relationship  between  these 

*  Public  Health,  1899-1900,  vol.  xii.,  pp.  139-213. 
f  Annual  Report  on  Health  of  Brighton,  1902,  p.  48 


SUPPURATION  311 

meteorological  conditions  and  milk,  though  an  open  question,  may 
be  an  essential  one  to  the  origin  of  the  disease.  Milk  is  probably 
a  common  vehicle  of  infection  (Ballard,  Delepine,  Newsholme),  and 
a  number  of  outbreaks  are  now  on  record  which  appear  to  have  been 
due  to  the  consumption  of  contaminated  milk.  In  1892  Gaffky 
recorded  an  outbreak  at  Giessen,*  in  1894  Mven  reported  on  160 
cases  of  diarrhoea  at  Manchester,-)-  in  1895  and  1898  three  outbreaks 
occurred  at  St  Bartholomew's  Hospital.]: 

The  facts  set  forth  above  furnish  sufficient  indication  of  the 
appropriate  methods  of  prevention. 

Suppuration  and  Abscess  Formation 

The  term  suppuration  is  used  to  designate  that  general  breaking 
down  of  cells  which  follows  acute  inflammation.  An  "abscess" 
is  a  collection,  greater  or  smaller,  of  the  products  of  suppuration, 
pus.  Pus  consists  chiefly  of  two  kinds  of  cells.  First,  leucocytes, 
which  have  immigrated  to  the  part  affected;  and  secondly,  broken 
clown  and  necrosed  elements.  Such  an  advanced  inflammatory 
condition  may  occur  in  any  locality  of  the  body,  and  it  will 
assume  different  characters  according  to  its  site.  There  are 
connected  with  suppuration,  as  causal  agents,  a  variety  of  bacteria. 
Pus  is  not  matter  containing  a  pure  culture  of  any  specific  species, 
but,  on  the  contrary,  is  generally  filled  with  a  large  number  of 
different  species,  each  playing  a  greater  or  lesser  part  in  the  process. 
The  most  important  are  as  follows  : — 

1.  The  Staphylococcus  group. — This  species  consists  of  micrococci 
arranged  in  groups,  which  have  been  likened  to  bunches  of  grapes 
(Plate  30,  p.  398).  They  are  the  common  organisms  found  in  pus,  and 
were,  with  other  auxiliary  bacteria,  first  distinguished  as  such  by  Pro- 
fessor Ogston  of  Aberdeen.  There  are  several  forms  of  the  same  species, 
differing  from  each  other  in  certain  respects.  Thus  we  have  the  8. 
pyogenes  aufeus  (golden-yellow),  albus  (white),  citreus  (lemon),  and 
others.  They  occur  commonly  in  nature,  in  air,  soil,  water,  as  well  as 
on  the  surface  of  the  skin,  and  in  all  suppurative  conditions.  The 
aureus  is  the  only  one  credited  with  pathogenic  virulence.  It  occurs 
in  the  blood  in  blood-poisoning  (septicaemia,  pyaemia),  and  is  present 
in  all  ulcerative  conditions,  including  ulcerative  disease  of  the  valves 
of  the  heart.  The  Staphylococcus  cereus  albus  and  S.  cereus  flavus  are 
slightly  modified  forms  of  the  8.  pyogenes  aureus,  and  are  differenti- 
ated from  it  by  the  fact  of  their  being  non-liquefying.  They  produce 
a  wax-like  growth  on  gelatine. 

*  Deut.  Med.  Woch.,  vol.  xviii.  p.  14. 

f  Annual  Report  Medical  Officer  of  Health,  Manchester,  1894. 
£  Report  of  Medical  Officer  of  Local  Government  Board,  1895-96,  pp.   197-204 ; 
ibid.,  1897-98,  p.  235  ;  and  ibid.,  1898-99,  p.  336. 


312 


BACTERIA  AND  DISEASE 


StaphylococciiA  pyogencs  aureus,  the  type  of  the  species,  is  grown 
in  the  laboratory  on  all  ordinary  media  at  room  temperature,  though 
more  rapidly  at  37°  C.  Liquefaction  sets  in  at  a  comparatively  early 
stage,  and  subsequently  we  have  in  gelatine  test-tube  cultures  a 
flocculent  deposit  of  a  bright  yellow  amorphous  mass,  and  in  gelatine 
plates  small  depressions  of  liquefaction  with  a  yellow  deposit.  The 
organism  renders  all  media  acid,  and  coagulates  milk.  Its  thermal 
death-point  in  gelatine  is  58°  C.  for  ten  minutes,  but  when  dry  con- 
siderably higher.  Outside  the  body  it  may  retain  vitality  for 


FIG.  25. — Diagram  of  Types  of  Streptococci. 

months.  It  stains  by  Gram's  method.  It  is  a  non-motile  and  a 
facultative  anaerobe;  but  the  presence  of  oxygen  is  necessary  for 
the  production  of  much  pigment.  Its  virulence  readily  declines. 

2.  Streptococcus  pyogenes. — In  this  species  of  micrococcus  the 
elements  are  arranged  in  chains.  Most  of  the  streptococci  in  pus, 
from  different  sources,  are  probably  of  one  species,  having  approxi- 
mately the  same  morphological  and  biological  characters.  Their 
different  effects  are  due  to  difierent  degrees  of  toxic  virulence ;  they 
are  generally  more  virulent  when  associated  with  other  bacteria,  for 
example,  the  Proteus  family. 

The  chains  vary  in  length,  consisting  of   more  elements  when 


SUPPURATION  313 

cultured  in  fluid  media  (hence  S.  longus  and  S.  brews).  They 
multiply  by  direct  division  of  the  individual  elements,  and  in  old 
cultures  it  has  been  observed  that  the  cocci  vary  in  form  and  size 
(involution  forms).  This  latter  fact  gave  support  to  the  theory  that 
streptococcus  reproduced  itself  by  arthrospores,  or  "  mother-cells." 

In  culture  upon  the  ordinary  media,  Streptococcus  pyogenes  is  com- 
paratively slow-growing,  producing  minute  white  colonies  on  or  / 
about  the  sixth  day.  It  does  not  liquefy  gelatine,  and  remains 
strictly  localised  to  the  track  of  the  inoculating  needle.  Like  the 
staphylococcus,  it  readily  loses  virulence.  The  thermal  death-point 
is,  however,  lower,  being  54°  C.  for  ten  minutes.  Marmorek  has 
devised  a  method  by  which  the  virulence  may  be  greatly  increased, 
and  he  holds  that  it  depends  upon  the  degree  of  virulence  possessed 
by  any  particular  streptococcus  as  to  what  effects  it  will  produce. 
By  the  adoption  of  Marmorek's  methods,  attempts  have  been  made  to 
prepare  an  antitoxin. 

Streptococcus  pyogenes  has  been  isolated  from  the  membrane  in 
cases  of  diphtheria,  and  from  small-pox,  scarlet  fever,  vaccinia,  and 
other  diseases.  In  such  cases  it  is  probably  not  the  causal  agent,  but 
merely  associated  with  the  complications  of  these  diseases.  Suppura- 
tion and  erysipelas  are  the  only  pathological  conditions  in  which  the 
causal  agency  of  Streptococcus  has  been  sufficiently  established. 

3.  The  Bacillus  pyocyaneus  occurs  in  green  pus,  and  is  the  cause 
of  the  coloration.     G-essard  was  the  first  to  prove  its  significance, 
and  he  described  two  varieties.     It  is  a  minute,  actively  motile,  non- 
sporulating  bacillus,  which  occasionally  complicates  suppuration  and 
produces  blue-green  pus.     It  stains  with  the  ordinary  aniline  stains, 
but   is   decolorised   by   Gram's   method.     Oxygen  is  necessary   for 
pigmentation,  which  is  due  to  two  substances :  pyocyanin,  a  greenish- 
blue  product  extracted  with  chloroform,  and  pyoxantJiosc,  a  brown 
substance  derived  from  the  oxidation  of  the  former  pigment.     Both 
these  colours   are  produced  in   cultivation   outside   the   body.     On 
gelatine  the  colour  produced  is  green,  passing  on  to  olive.     There  is 
liquefaction.     On  potato  we  generally  obtain  a  brown  growth  (com- 
pare B.  coli,  B.  mallei,  and  others).     The  ..    ,. 
organism  grows  rapidly  on  all  the  ordinary                 •::/     "*lg*       Jf 
media,  which  it  has  a  tendency  to  colour       »«u          ** 
throughout.     It-  will  be  remembered  that           "*  /,  •' 

when  speaking  of  the  antagonism  of  organ-  /f 

isms,  we  referred  to  the  inimical  action  of 

B.  pyocyaneus  upon  the  bacillus  of  anthrax.  ft       ^ 

4.  MicrOCOCCUS  tetragonUS. This  Species       FIG.  26.— Diagram  of  Micrococcus 

occurs  in  phthisical  cavities,  and  in  certain 

suppurations  in  the  region  of  the  face.     The  micrococcus  usually 

occurs  in  the  form  of  small  tetrads.     A  capsule  is  generally  present. 


314  BACTERIA  AND  DISEASE 

It  is  a  non-liquefying  organism,  pathogenic  for  white  mice  (producing 
septicsemia).  It  grows  on  ordinary  laboratory  media,  producing  a 
viscid  tenacious  culture. 

5.  B.  coli  communis  and   many   putrefactive   germs  commonly 
occur  in  suppurative  conditions,  but  they  are  not  restricted  to  such 
disorders  (see  p.  46). 

6.  Micrococcus    gonorrhcece    (Neisser,    1879). — This   organism    is 
more  frequently  spoken  of  as  a  diplococcus.     It  occurs  at  the  acute 
stage  of  the  disease  (and  in  the  purulent  secretion  of  gonorrhoeal 
conjunctivitis),  but  is  not  readily  differentiated  from  other  similar 
diplococci  except  by  laboratory  methods.     Each  element  of  the  diplo- 
coccus presents  a  straight  or  concave  surface  to  its  fellow.     A  very 
marked  concavity  indicates  commencing  fission.     The  position  which 
these  diplococci  take  up  in  pus  is  intracellular,  and  they  are  arranged 
more  or  less  definitely  around  the  nucleus.     In  chronic  gonorrhoea 

the  diplococci  are  diminished  in  number. 
Difficulty  has  often  been  found  in  culti- 
vating the  organism  in  artificial  media 
outside  the  body.  Wertheim  and  others 
have  suggested  special  formulae  for  the 
preparation  of  suitable  media,  but  it  is  a 
comparatively  simple  matter  to  secure 
cultures  on  agar  plates  smeared  with 
human  blood  from  a  pricked  finger.  The 
plate  is  incubated  at  37°  C.  At  the  end 
of  twenty-four  hours  small  raised  grey 
colonies  appear,  which  at  about  the  end 

FiG.27.-DiagramofGouococcus.       of  f™r  days  show  adult  growth.     The 

margin  is  uneven,  and  the  centre  more 

opaque  than  the  rest  of  the  colony.  This  diplococcus  is  readily 
killed,  and  sub-cultures  must  be  frequently  made  to  retain  vitality 
and  virulence.  Light,  desiccation,  and  a  temperature  of  55°  C. 
all  act  germicidally.  The  organism  stains  readily  with  Lofner's  blue, 
but  is  decolorised  by  Gram's  method.  It  is  more  or  less  strictly 
parasitic  to  man,  and  has  been  definitely  proved  to  be  the  cause 
of  gonorrhoea.  A  toxin  has  been  separated.  The  shape,  size, 
character  of  growth,  intracellular  position,  and  staining  pro- 
perties of  the  gonococcus  assist  in  differentiating  it  from  various 
similar  diplococci.*  An  organism  not  greatly  different  from  the 
gonococcus  is  the  diplococcus  intracellularis  meningitidis  isolated  by 
Weichselbaum  from  cases  of  cerebro-spinal  meningitis.  It  occurs  in 
the  interior  of  leucocytes. 

Such  are  the  chief  organisms  associated  with  suppuration.     In 

the  condition  known  as  septicaemia,  these  organisms  multiply  in  the 

*  See  Tram.  Jmner  Inst.  (First  Series),  A.  G.  R.  Foulerton,  F.R.C.S.,  pp.  40-81. 


ANTHRAX  315 

blood,  and  give  rise  to  general  poisoning  without  abscess  formation ; 
in  pyaemia,  however,  multiple  abscesses  occur  in  various  parts  of  the 
body,  including  internal  organs.  Erom  the  results  of  experiment  it  is 
now  believed  that  suppuration  in  any  form  or  degree  is  invariably 
the  result  of  bacterial  infection.  But  it  is  not  known  in  what  way 
bacteria  exactly  cause  the  condition ;  it  may  be  due  to  extracellular 
toxins,  or  intracellular  poisons,  or  to  the  bodies  themselves  setting  up 
primary  irritation,  or  to  all  three  conditions.  Positive  cliemiotaxis  is 
probably  the  explanation  of  the  immigration  of  the  leucocytes. 


Anthrax 

This  disease  was  one  of  the  first  in  which  the  causal  agency  of 
bacteria  was  proved.  In  1849  Pollender  found  an  innumerable 
number  of  small  rods  in  the  blood  of  animals  suffering  from  anthrax. 
In  1863  Davaine  described  these,  and  attributed  the  disease  to  them. 
But  it  was  not  till  1876  that  Koch  finally  settled  the  matter  by 
isolating  the  bacilli  in  pure  culture  and  describing  their  biological 
characters. 

It  is  owing  in  part  to  its  interesting  bacterial  history,  which 
opened  up  so  much  new  ground  in  this  comparatively  new  science, 
that  anthrax  has  assumed  such  an  important  place  in  pathology. 
But  for  other  reasons,  too,  it  claims  attention.  It  appears  to  have 
been  known  in  the  time  of  Moses,  and  was  perhaps  the  disease 
described  by  Homer  in  the  First  Book  of  the  Iliad.  Eome  was 
visited  by  it  in  740  B.C. 

Anthrax  is  an  acute  disease,  affecting  sheep,  cattle,  horses,  goats, 
deer,  and  man.  Cats,  white  rats,  and  Algerian  sheep  are  immune. 
Swine  become  infected  by  feeding  on  the  offal  of  diseased  cattle 
(Crookshank). 

Clinical  Characters. — In  most  instances  the  first  intimation  of  an  outbreak  of 
anthrax  is  the  discovery  of  a  dead  animal  in  the  pasture  or  byre.  The  animal  may 
have  been  left  a  few  hours  earlier  in  apparent  good  health  ;  at  least,  there  may  have 
been  nothing  to  attract  attention,  or  give  warning  of  the  near  approach  of  death. 
Occasionally  there  are,  however,  premonitory  symptoms  of  an  attack  of  anthrax 
which  can  be  recognised  by  an  expert.  The  affected  animal  is  dull,  and  disinclined 
to  move.  If  the  case  occurs  in  a  herd  at  pasture  the  fact  is  sometimes  indicated  by 
the  separation  of  the  sick  animal  from  the  rest.  The  affected  animal  will  occasionally 
cease  to  feed,  and  stand  with  its  head  bent  towards  the  ground,  and  sometimes  a 
little  blood  is  discharged  from  the  nostrils  and  also  with  the  faeces.  Close  attention 
will  enable  the  observer  to  detect  an  occasional  shiver  and  trembling  of  the  limbs, 
which  passes  rapidly  over  the  body,  and  then  ceases.  The  shivering  fits  may  then 
become  more  frequent,  and  perhaps,  while  these  signs  are  being  noted,  the  animal 
will  suddenly  roll  over  on  its  side,  and,  after  a  few  violent  struggles,  expire.  On 
close  inspection,  especially  in  the  case  of  swine,  it  will  often  be  found  that  there  is  a 
good  deal  of  swelling  under  the  throat  extending  down  the  neck ;  and  the  swollen 
part  will  at  first  be  hot  and  tender  to  the  touch,  but  as  the  disease  progresses 
it  becomes  insensitive  and  cold. 


316 


BACTERIA  AND  DISEASE 


The  post-mortem  signs  are  mainly  three:  The  spleen -is  greatly 
enlarged  and  congested,  is  dark  red  in  colour,  friable  to  the  touch, 
and  contains  enormous  numbers  of  bacilli ; '  the  skin  may  show 
exudations  forming  dark  gelatinous  tumours ;  and  the  blood  remains 
fluid  for  some  time  after  death,  is  black  and  tar-like,  contains  bubbles  of 
air,  and  shows  other  degenerative  changes  in  the  red  corpuscles,  whilst 
the  small  Uood-vessels  contain  such  vast  quantities  of  bacilli  that  they 
may  be  ruptured  by  them.  Particularly  is  this  true  in  the  peripheral 
arteries.  Many  of  the  organs  of  the  body  show  marked  congestion. 

The  bacilli  of  anthrax  are  square-ended  rods  1  yu  broad  and 
4-5  IUL  long.  In  the  tissues  of  the  body  they  follow  the  lines  of  the 
capillaries,  and  are  irregularly  situated.  In  places  they  are  so 
densely  packed  as  to  form  obstructions  to  the  onward  flow  of  blood. 
In  cultures  they  occur  in  chains  end  to  end,  having,  as  a  rule,  equal 

interbacillary  spaces.  But  long  filaments 
and  threads  also  occur.  The  exact  shape 
of  the  bacillus  depends,  however,  upon 
staining  and  spore  formation.  Both 
these  factors  may  very  materially  modify 
the  normal  shape.  The  spores  of  anthrax 
are  oval  endospores,  produced  only  in 
the  presence  of  free  oxygen,  and  at  any 
temperature  between  18  and  41°  C.  On 
account  of  requiring  free  oxygen,  they 
are  formed  only  outside  the  body.  The 
homogeneous  protoplasm  of  the  bacillus 
becomes  granular ;  the  granules  coalesce, 
constituting  spores.  Each  spore  pos- 
sesses a  thick  capsule,  which  enables  it  to  resist  many  physical 
conditions  which  kill  the  bacillus.  When  the  spore  is  ripe,  or 
has  exhausted  the  parent  bacillus,  it  may  either  take  on  a  resting 
stage,  or  under  favourable  circumstances  commence  germination, 
very  much  after  the  manner  of  a  seed.  The  spores  may  infect 
a  farm  for  many  months;  indeed,  cases  are  on  record  which 
appear  to  prove  that  the  disease  on  a  farm  in  the  autumn  may, 
by  means  of  the  spores,  be  carried  on  by  the  hay  of  the  follow- 
ing summer  into  a  second  winter.  Thus,  by  means  of  the  spores, 
the  infection  of  anthrax  may  cling  to  the  land  for  very  long  periods, 
even  for  years.  Spores  of  anthrax  can  withstand  5  per  cent,  carbolic 
acid  or  1-1000  corrosive  sublimate  for  more  than  an  hour;  even 
boiling  does  not  kill  them  at  once,  whilst  the  bacilli  without  their 
spores  are  killed  at  54°  0.  in  ten  minutes.  When  the  spores  are  dry 
they  are  much  more  resistant  than  when  moist.  The  persistence  of 
the  anthrax  bacillus  is  due  to  its  spores. 

The    bacillus    is    aerobic,   non-motile,   and    liquefying.      Broth 


FIG.  28.— Diagram  of  Bacillus  of 
Anthrax  and  Blood  Corpuscles. 


ANTHRAX  317 

cultures  become  turbid  in  thirty-six  hours,  with  nebulous  masses  of 
threads  matted  together.  The  pellicle  which  forms  on  the  surfaces 
affords  an  ideal  place  for  spore  formation.  Cultures  in  the  depth 
of  gelatine  show  a  most  characteristic  growth.  From  the  line  of 
inoculation  delicate  threads  and  fibrillse  extend  outwards  horizontally 
into  the  medium.  Liquefaction  commences  at  the  top,  and  eventually 
extends  throughout  the  tube.  On  gelatine  plates  small  colonies 
appear  in  thirty-six  hours,  and  on  the  second  or  third  day  they 
appear,  under  a  low  power  of  the  microscope,  not  unlike  matted 
hair.  The  colonies  after  a  time  sink  in  the  gelatine,  owing  to  lique- 
faction. On  potato,  agar,  and  blood  serum  the  anthrax  bacillus 
grows  well  (Plates  17  and  22). 

Channels  of  Infection.  1.  The  Alimentary  Canal. — This  is  the 
usual  mode  of  infection  in  animals  grazing  on  infected  pasture  land. 
A  soil  suitable  for  the  propagation  of  anthrax  is  one  containing 
abundance  of  air  and  proteid  material.  Feeding  on  bacilli  alone  might 
possibly  not  produce  the  disease,  owing  to  the  germicidal  effect  of 
the  gastric  juice.  But  spores  can  readily  pass  uninjured  through 
the  stomach,  and  produce  anthrax  in  the  blood.  Infected  water,  as 
well  as  fodder,  may  convey  the  disease.  Water  becomes  infected  by 
bodies  of  animals  dead  of  anthrax,  or,  as  was  the  case  once  at  least 
in  the  south-west  of  England,  by  a  stream  passing  through  the 
washing-yard  of  an  infected  tannery.  Manure  on  fields,  litter  in 
stalls,  and  infected  earth,  may  all  contribute  to  the  transmission  of 
the  disease.  Darwin  pointed  out  the  services  which  are  performed 
in  superficial  soils  by  earthworms  bringing  up  casts ;  Pasteur  was  of 
opinion  that  in  this  way  earthworms  were  responsible  for  continually 
bringing  up  the  spores  of  the  anthrax  bacillus  from  buried  corpses 
to  the  surface,  where  they  would  reinfect  cattle.  Koch  disputed  this, 
but  more  recently  Bellinger  has  demonstrated  the  correctness  of 
Pasteur's  views  by  isolating  anthrax  contagium  from  5  per  cent,  of 
the  worms  sent  him  from  an  anthrax  pasture.  Bollinger  also 
maintains  that  flies  and  other  insects  may  convey  the  disease  from 
discharges  or  carcases  round  which  they  congregate. 

Alimentary  infection  in  man  is  a  rare  form,  and  it  reveals  itself 
in  a  primary  diseased  state  known  as  mycosis  intestinalis,  an  inflamed 
condition  of  the  intestine  and  mesenteric  lymph-glands. 

2.  Through  the  Skin. — Cutaneous  anthrax,  when  it  occurs  in  the 
human  subject,  goes  by  the  name  of  malignant  pustule,  and  is  caused 
by  infective  anthrax  matter  gaining  entrance  through  abrasions  or 
ulcers  in  the  skin.  This  local  form  is  obviously  mostly  contracted  by 
those  whose  occupation  leads  them  to  handle  hides  or  other  anthrax 
material  (butchers  and  cleaners  of  hides),  and  it  naturally  affects  the 
skin  of  the  hands,  forearms,  face,  or  back  (as  it  occurs  amongst  hide- 
porters).  Two  or  three  days  after  inoculation  a  red  pimple  appears, 


318  BACTERIA  AND  DISEASE 

which  rapidly  passes  through  a  vesicular  stage  until  it  is  a  pustule. 
Concomitantly,  we  have  glandular  enlargement  (the  pustule  acting 
as  a  centre  of  subcutaneous  oedema),  general  malaise,  and  a  high 
temperature.  Thus  from  a  local  sore  a  general  infection  may  result. 
Unless  this  does  occur,  the  issue  is  not  likely  to  he  fatal,  and  the 
bacilli  will  not  gain  entrance  into  the  blood.  The  spleen  is  usually 
not  affected,  and  the  organs  generally  contain  few  or  no  bacilli. 
When  a  fatal  issue  occurs,  it  is  due  to  the  absorption  of  toxins. 
Early  excision  of  the  pustule  is  usually  followed  by  recovery.* 

3.  Respiratory  Tract. — In  man,  this  is  perhaps  the  commonest 
form  of  all,  and  is  well  known  under  the  term  "  wool-sorters'  disease," 
or  pulmonary  anthrax.  This  mode  of  infection  occurs  when  dried 
spores  are  inhaled  in  processes  of  skin-cleaning.  It  frequently  com- 
mences as  a  local  lesion,  affecting  the  mucous  membrane  of  the 
trachea  or  bronchi,  but  it  rapidly  spreads,  affecting  the  neighbouring 
glands,  which  become  greatly  enlarged,  and  extending  to  the  pleura 
and  lung  itself.  The  lung  shows  collapse  and  oedema  leading  to 
pulmonary  embarrassment.  There  is  also  fever.  Such  cases,  as  a 
rule,  rapidly  end  fatally.  Even  in  wool-sorters'  disease  the  bacilli 
do  not  become  widely  distributed. 

Preventive  Methods. — As  a  rule,  anthrax  carcases  are  better 
not  opened  and  exposed  to  free  oxygen.  An  extended  post- 
mortem examination  is  not  necessary.  A  small  prick,  for  example, 
in  the  auricular  vein  will  extract  enough  blood  to  examine  for 
the  anthrax  bacilli,  which  are  driven  by  the  force  of  the  blood 
current  to  the  small  surface  capillaries.  This  occurs,  of  course, 
only  when  the  disease  has  become  quite  general,  for  in  the  early 
stage  the  healthy  blood  limits  the  bacilli  to  the  internal  organs. 
In  such  cases  examination  of  the  blood  of  the  spleen  is  necessary. 
The  chief  source  of  danger  is  the  infection  by  anthrax  Blood  or 
discharges  (containing  sporulating  bacilli)  of  the  field,  farm-yard, 
byres,  etc.,  and  it  is  therefore  necessary  for  thorough  disinfection  to 
be  carried  out  if  infection  has  occurred.  Burning  the  entire  carcase 
in  a  crematorium  would  be  the  ideal  treatment.  As  such  is  not 
generally  feasible,  the  next  best  thing  is  to  bury  the  carcase  deeply 
with  lime  below  and  above  it,  and  rail  in  the  area  to  prevent  other 
animals  grazing  off  it. 

In  the  German  Special  Eules  relating  to  the  establishment  and 
management  of  horse-hair  spinning-mills,  factories  for  hair  and  bristle 
dressing,  and  brush  factories  of  all  kinds,f  it  is  laid  down  that 
disinfection  may  be  done  in  one  of  the  three  following  ways :  (1)  by 

*  Accidental  infection  with  anthrax  has  been  held  to  be  an  accident  to  employes 
under  the  Workmen's  Compensation  Act,  1897  (Courts  of  Appeal),  Justice  of  the 
Peace,  May  7,  1904,  vol.  Ixviii.,  p.  193. 

t  Order  dated  October  22,  1902,  under  Industrial  Code  (Qewerbeordnuny),  120e. 


PLATE  22. 


Bacillus  an,tkradt>. 
Gelatine  stab  culture.    3  days'  growth  at  20°  C      x  I£  times. 


Jjacillus  anthracis. 

Smear  preparation  from  splenic  blood  of  cow. 
x  1000. 


FRANKEL'S  PNEUMOCOCCUS  IN  PNEUMONIC  SPUTUM. 
Stained  with  Neelsen  and  methylene  blue. 

x    1000. 


[To  face  page  318. 


PNEUMONIA  319 

letting  a  current  of  steam  act  on  the  material  for  not  less  than  half 
an  hour,  at  a  temperature  of  218°  F. ;  (2)  by  boiling  for  not  less  than 
one  hour  in  a  solution  containing  2  per  cent,  potassium  permanganate, 
bleaching  it  afterwards  with  a  solution  containing  3  to  4  per  cent,  of 
sulphurous  acid ;  or  (3)  by  boiling  in  water  for  not  less  than  two 
hours.  A  number  of  other  regulations  are  included  in  the  Order.* 
Various  experiments  have  been  carried  out  in  this  country  with  a 
view  to  determining  the  most  effectual  methods  of  disinfection.-)- 
Boiling  does  not  appear  to  be  always  effective,  and  is,  moreover, 
frequently  impracticable  owing  to  the  damage  it  causes.  In  steam 
disinfection  of  horse  hair  and  similar  materials,  a  temperature  of 
230°  F.  for  30  minutes  is  as  effective  as  higher  temperatures,  but  the 
hair  must  be  loose  and  not  closely  packed  in  bundles.  Probably  one 
of  the  most  practical  methods  for  disinfection  of  hair  is  to  soak  it 
for  twenty-four  hours  in  a  solution  of  one  part  of  corrosive  sublimate 
in  a  thousand  parts  of  warm  water  (Klein).  But  apart  from  actual 
disinfection  of  the  material,  considerable  protection  is  afforded  by 
(a)  the  avoidance  of  horse  hair  from  Eussia,  Siberia,  and  China,  and 
wool  from  Persia  (from  which  sources  most  infection  is  derived),  unless 
it  is  guaranteed  as  thoroughly  disinfected ;  (b)  by  compelling  employes 
to  wash  with  soap  and  hot  water  before  leaving  work  or  taking  food, 
the  more  general  the  washing,  as  a  rule,  the  greater  the  security 
obtained ;  (c)  the  use  of  fans  creating  a  down-draught  to  remove  dust 
when  sorting;  and  (d)  the  exclusion  of  workpeople  suffering  from 
cuts  or  scratches  of  the  skin  from  processes  in  which  they  are  likely 
to  come  into  contact  with  dust  from  horse  hair. 

Anthrax  covers  a  wide  geographical  area  all  over  the  world,  and 
no  country  seems  altogether  exempt.  In  Germany  as  many  as  3700 
animals  have  been  lost  in  a  single  year.  In  1903  there  were  761 
outbreaks  of  anthrax  in  Great  Britain,  in  which  1127  animals  were 
attacked.  This  is  the  largest  return  recorded  since  the  passing  of 
the  Anthrax  Order  in  1886. 

Pneumonia 

Some  of  the  difficulty  which  has  surrounded  the  bacteriology 
of  inflammation  of  the  lungs  is  due  to  the  confusion  arising 
from  supposing  that  attacks  of  the  disease  differed  only  in 
degree.  Pneumonia,  however,  has  various  forms,  arising  now  from 
one  cause,  now  from  another.  The  lobar  or  croupous  pneumonia  is 
associated  with  two  organisms :  Frankel's  diplococcus  and  Fried- 
lander's  pneumo-bacillus.  Acute  catarrhal  pneumonia  generally 
arises  as  a  secondary  complication  to  other  disease,  such  as  diphtheria, 
influenza,  bronchial  affections,  etc.  Septic  pneumonias  are  also  not 

*  Annual  Report  of  Chief  Inspector  of  Factories  and  Workshops^  1902,  p.  214. 
t  Md.,  1900,  1902,  and  1903. 


320  BACTERIA  AND  DISEASE 

specific,  but  secondary  or  mechanical.  Other  bacteria  in  addition 
to  the  two  named  have  from  time  to  time  been  held  responsible 
for  pneumonia,  a  streptococcus  receiving,  at  one  time,  some  support. 
But  whilst  opinion  is  divided  as  to  the  rdle  of  various  extraneous 
and  concomitant  bacteria  in  lung  disease,  importance  is  attached  to 
Frankel's  and  Friedlander's  organisms. 

The  diplococcus  of  Frankel  is  a  small  oval  diplococcus  found  in 
the  "rusty"  sputum  of  croupous  pneumonia.  It  is  non-motile, 
non-liquefying,  aerobic,  and  facultatively  anaerobic.  When  examined 
from  cultures  the  diplococci  are  frequently  seen  in  chains,  not  unlike 
a  streptococcus,  and  there  is  some  reason  to  suppose  that  this  form 
gave  rise  to  the  belief  that  it  was  another  species ;  when  examined 
from  the  tissues,  sputum,  or  pus,  it  possesses  a  capsule,  like  an 
unstained  halo  (stained  by  MacConkey's  method),  but  in  culture  this 
is  lost  except  in  gelatine  at  37°  C.  (Gordon).  Involution  forms  occur. 
The  diplococcus  is  difficult  to  cultivate,  but  grows  on  glycerine 
agar  and  blood  serum  at  body  temperature.  .  On  ordinary  gelatine 
at  room  temperature  it  does  not  grow,  or  if  so,  very  slightly.  The 
ideal  fluid  is  a  slightly  alkaline  liquid  medium,  and  in  twenty-four 
hours  a  powdery  growth  will  occur  in  such  broth.  On  potato  there 
is  apparently  no  growth.  The  pneumococcus  always  requires  a 
temperature  of  about  blood-heat  for  its  maximum  development. 
It  rapidly  loses  its  virulence  on  solid  media,  and  is  said  to  be 
non-virulent  after  three  or  four  sub-culturings.  A  temperature  of 
54-58°  C.  for  a  few  minutes  kills  the  bacteria,  but  not  the  toxin. 
This,  however,  is  removed  by  filtration,  and  is  therefore  probably 
intracellular.  It  is  attenuated  by  heating  to  70°  C.  This  diplococcus 
stains  by  Gram's  method  (see  Plate  22,  p.  318). 

Frankel's  diplococcus  occurs,  then,  in  the  acute  stage  of  true 
croupous  pneumonia,  in  company  with  streptococci  and  staphylococci. 
It  is  by  far  the  most  frequently  present  organism  in  croupous 
pneumonia.  It  also  occurs  in  the  blood  in  certain  suppurative 
conditions,  in  pleurisy  and  inflammation  of  the  pericardium,  and  some- 
times in  diphtheria,  and  therefore  it  is  not  peculiar  to  pneumonia. 

Frankel's  organism  is  said  to  be  frequently  present  in  the  saliva 
of  healthy  persons.  Inflammation  depresses  the  resistant  vitality 
of  the  tissues,  and  thus  affords  to  the  diplococcus  present  in  the 
saliva  an  excellent  nidus  for  its  growth.* 

Friedlander's  Pneumo-bacillus  is  a  capsulated  oval  coccus,  assuming 
the  form  of  a  small  bacillus.  It  is  inconstant  in  pneumonia,  unequally 
distributed,  and  scarce ;  it  is  aerobic,  and  facultatively  anaerobic ; 

*  For  further  particulars  respecting  the  pneumococcus,  see  Practitioner,  March 
1900,  pp.  280-304  (J.  W.  Eyre);  and  Brit.  Med.  Jour.,  1902,  vol.  ii.,  pp.  1585, 
1646,  1704,  1765  (Croonian  Lectures  on  Natural  History  and  Pathology  of 
Pneumonia,  by  J.  W.  Washbourn). 


ACtlNOMYCOSiS  321 

it  occasionally  occurs  in  long  forms  and  filaments ;  it  is  non-motile, 
non-liquefying,  and  has  no  spores;  it  does  not  stain  by  Gram's 
method,  which  stain  is  therefore  used  for  differential  diagnosis ;  it  will 
grow  fairly  well  in  ordinary  gelatine  at  20°  C. ;  and  it  is  a  denitrifying 
organism,  and  also  an  actively  fermentative  one,  even  fermenting 
glycerine.  It  is  not  unlike  B.  coli  communis,  and  to  distinguish  it  from 
that  organism  it  should  be  remembered  that  the  B.  coli  is  motile, 
never  has  a  capsule,  produces  indol,  and  does  not  ferment  glycerine. 

It  is  now  generally  held  that  Frankel's  diplococcus  is  the  chief 
factor  in  the  causation  of  croupous  pneumonia,  and  probably  plays 
an  important  part  in  other  forms  of  the  disease.  In  the  septic 
pneumonias  the  different  suppurative  organisms  are  found,  and  some- 
times in  ordinary  pneumonias  these  organisms  may  be  the  causal 
agents. 

Influenza 

In  1892,  during  the  pandemic  of  influenza,  Pfeiffer  dis- 
covered a  bacillus  in  the  bronchial  mucus  of  patients  suffering 
from  the  disease.  It  is  one  of  the  smallest  bacilli  known,  and 
frequently  occurs  in  chains  not  unlike  a  streptococcus.  Canon 
obtained  the  same  organism  from  the  blood.  In  the  bronchial 
expectoration  it  can  retain  its  virulence  for  as  long  as  a  fortnight, 
but  it  is  quickly  destroyed  by  drying.  The  bacillus  is  aerobic,  non- 
rnotile,  and  up  to  the  present  spores  have  not  been  found.  It  is 
non-motile,  and  does  not  stain  by  Gram's  method.  It  has  no 
capsule.  It  grows  somewhat  feebly  in  artificial  media,  and  readily 
dies  out.  Blood  serum,  glycerine  agar,  blood  agar,  and  gelatine  have 
all  been  used  at  blood-heat.  It  does  not  grow  at  room  temperature. 
On  blood  agar  colonies  appear  in  twenty-four  hours  in  the  form 
of  minute  circular  dots,  almost  transparent.  The  bacilli  die  out 
quickly  in  cultures.  Pfeiffer's  bacillus  appears  most  abundantly  at 
the  height  of  the  disease,  and  disappears  with  convalescence.  It  is 
said  not  to  appear  in  any  other  disease.  It  is  chiefly  found  in  the 
respiratory  passages  in  cases  of  influenza,  and  is  usually  isolated  from 
nasal  secretion  and  the  masses  of  greenish-yellow  bronchial  sputum. 
The  bacilli  may  persist  after  recovery  of  the  patient. 

Actinomycosis 

This  disease  affects  both  animals  and  man.  As  Professor 
Crookshank  has  pointed  out,  it  has  long  been  known  in  this 
country,  but  its  various  manifestations  have  been  mistaken  for 
other  diseases  or  have  received  popular  names.* 

*  Bacteriology  and  Infective  'Diseases  (1896),  pp.  413-447.  Professor  Crookshank's 
Reports  to  the  Agricultural  Department  of  the  Privy  Council  constitute  a  most 
complete  account  of  this  disease.  See  also  Trans.  Jenner  Institute  (Second  Series), 
1899,  p.  17. 

X 


322  BACTERIA  AND  DISEASE 

Here  mention  can  only  be  made  of  the  most  outstanding  facts 
concerning  the  disease.  It  is  caused  by  the  "ray  fungus,"  or 
Streptothrix  actinomyccs,  one  of  the  higher  bacteria  which,  growing 
on  certain  cereals,  may  gain  entrance  to  the  tissues  of  man  and  beast 
by  lacerations  of  the  mucous  membrane  of  the  mouth,  by  wounds,  or 
by  decayed  teeth.  Barley  has  been  the  cereal  in  question  in  some 
cases.  The  result  of  the  introduction  of  the  parasite  is  an  "  infective 
granuloma."  This  is,  generally  speaking,  of  the  nature  of  an  inflam- 
matory tumour  composed  of  round  cells,  epithelioid  cells,  giant  cells, 
and  fibrous  tissue,  forming  nodules  of  varying  sizes.  In  some  cases 
they  develop  to  large  tumours,  in  others  they  soon  break  down. 
Actinomycosis  resembles  tuberculosis  in  some  of  its  tissue  characters. 

In  the  discharge  or  pus  from  human  cases  of  the  disease  small 
sulphur-yellow  bodies  may  be  detected,  and  these  are  tufts  of  "  dubs  " 
which  are  the  broken-down  rays  of  the  parasite ;  for  in  the  tissues 
which  are  affected  the  parasite  arranges  itself  in  a  radiate  manner, 
growing  and  extending  at  its  outer  margin  and  degenerating  behind. 
In  cattle  the  centre  of  the  old  ray  becomes  caseated,  or  even  calcified. 
In  the  human  disease  abundant  "  threads "  are  formed  as  a  tangled 
mass  in  the  middle  of  the  colony.  As  clubs  characterise  the  bovine 
actinomycosis,  so  threads  are  the  feature  of  the  human  form  of  the 
disease.  But  in  both  there  is  a  third  element,  namely,  small  round 
cells,  called  by  some  spores,  by  others  simply  cocci.  They  are 
probably  formed  from  the  filaments,  but  authorities  are  not  yet 
agreed  as  to  the  precise  significance  and  role  of  these  round  cells. 
The  life-history  of  the  micro-organism  may  be  summed  up  thus : 
"The  spores  sprout  into  excessively  fine,  straight  or  sinuous,  and 
sometimes  distinctly  spirilliform,  threads,  which  branch  irregularly 
and  sometimes  dichotomously.  The  extremities  of  the  branches 
develop  the  club-shaped  bodies.  The  clubs  are  closely  packed 
together,  so  that  a  more  or  less  globular  body  is  formed,  with  a  central 
core  composed  of  a  dense  mass  of  threads  "  (Crookshank).  (Plate  13, 
p.  140.) 

In  man  the  disease  manifests  itself  in  various  parts  according 
to  the  point  of  entrance.  It  has  occurred  in  the  mouth,  vertebrae, 
oesophagus,  intestine,  liver,  kidneys,  lungs,  etc.  When  occurring  in 
the  mouth,  it  attacks  the  lower  jaw  most  frequently.  In  one  recorded 
case  the  disease  was  localised  to  the  bronchi,  and  did  not  even  extend 
into  the  lungs.  It  was  probably  contracted  by  inhalation  of  the 
parasite.  The  disease  may  spread  to  distant  parts  by  means  of  the 
blood  stream  (rnetastatic  abscesses),  and  frequently  the  abscesses  arc 
apt  to  burrow  in  various  directions.  The  chronic  inflammatory 
change  usually  ends  in  suppuration. 

In  the  ox  the  disease  remains  much  more  localised,  is  more 
formative,  and  frequently  occurs  in  the  lower  jaw,  palate,  or  tongue. 


GLANDERS  323 

In  the  latter  site  it  is  known  as  "wooden  tongue/'  owing  to  the 
hardness  resulting.  The  skin  and  subcutaneous  tissues  are  also  a 
favourite  seat  of  the  disease,  producing  the  so-called  wens  or  clyers 
so  commonly  seen  in  the  fen-country  (Crookshank).  Actinomycosis 
in  cattle  is  especially  prevalent  in  river  valleys,  marshes,  and  on 
land  reclaimed  from  the  sea.  The  disease  occurs  at  all  seasons,  but 
perhaps  more  commonly  in  autumn  and  winter.  It  is  more  frequently 
met  with  in  young  animals.  The  disease  is  probably  not  hereditary 
nor  readily  communicated  from  animal  to  animal. 

The  Streptothrix  Actinomyces  may  be  cultivated,  like  other 
parasites,  outside  the  body.  Gelatine,  blood  serum,  agar,  glycerine 
agar,  and  potato  have  been  used  for  this  purpose.  After  a  few  days  on 
glycerine  agar  at  the  temperature  of  the  blood,  small,  white,  shining 
colonies  appear,  which  increase  and  coalesce.  In  about  ten  days' 
time  the  culture  often  turns  a  bright  yellow,  though  it  may  remain 
white  or  even  take  on  a  brown  or  olive  tint.  The  entire  mass  of 
growth  is  raised,  dry,  corrugated,  and  crinkled,  and  composed  almost 
exclusively  of  threads.  In  its  early  stage  small  bacillary  forms 
occur,  and  in  its  later  stage  coccal  forms.  True  clubs  never  occur 
in  pure  cultures,  although  the  threads  may  occasionally  show  bulbous 
endings. 

Glanders 

Glanders  in  the  horse  and  ass,  and  sometimes  by  communication 
in  man  also,  is  caused  by  a  short,  non-motile,  aerobic  bacillus,  named, 
after  the  old  Roman  nomenclature  (malleus),  Bacillus  mallei.  It  was 
discovered  in  1882  by  Loffler  and  Schlitz.  It  is  found  in  the  nasal 
discharge  of  glandered  animals.  In  appearance,  the  bacillus  is  not 
unlike  B.  tuberculosis,  except  that  it  is  shorter  and  thicker.  The 
beading  of  the  bacillus  of  glanders,  like  that  in  tubercle,  does  not 
denote  spores.  B.  mallei  can  be  cultivated  on  the  usual  media, 
especially  on  glycerine  agar  and  potato.  On  the  last-named  medium 
at  blood-heat  it  forms  a  very  characteristic  honey-like  growth,  which 
later  becomes  reddish-brown.  High  temperature  is  usually  necessary. 

In  the  horse  glanders  may  affect  the  nasal  mucous  membrane, 
forming  nodules  which  degenerate  and  emit  an  offensive  discharge. 
From  the  nose,  or  nasal  septum,  as  a  centre,  the  disease  may  spread 
to  surrounding  parts.  It  may  also  occur  as  nodules  in  and  under 
the  skin,  and  involving  the  superficial  lymph  vessels  and  glands, 
when  it  is  known  as  "  farcy."  Persons  attending  a  glandered  animal 
may  contract  the  disease,  often  by  direct  inoculation.  Horned  cattle 
are  immune. 

In  man  glanders  occurs  in  two  forms,  an  acute  and  a  chronic. 
The  site  is,  of  course,  usually  on  the  hand  or  arm.  The  acute  form 
has  the  appearance  of  a  "poisoned  wound,"  locally,  and  there  are 


324  BACTERIA  AND  DISEASE 

also  the  general  symptoms  of  pyaemia,  and  an  eruption  on  the 
surface  of  the  body.  Such  cases  usually  terminate  fatally.  The 
chronic  form  results  in  local  ulceration  and  involvement  of  the 
lymphatics.  It  may  at  any  time  become  acute. 

The  glanders  bacillus  is  not  quickly  destroyed  by  drying,  but  it 
possesses  comparatively  feeble  resistance  to  heat  (55°  C.  for  ten 
minutes),  and  antiseptics  (5  per  cent,  carbolic  in  three  minutes).  It 
differs  widely  from  the  tubercle  bacillus  in  staining  properties. 
Gram's  method  and  that  of  Ziehl-Neelsen  are  inapplicable.  Carbol- 
thionin  blue  is  the  best  stain  to  use.  (Plate  13,  p.  140.) 

Mallein  is  a  substance  analogous  to  tuberculin,  and  is  made  by 
growing  a  pure  culture  of  B.  mallei  in  glycerine-veal  broth  in  flat 
flasks,  with  free  access  of  calcined  air.  After  a  month's  growth  the 
culture  is  sterilised,  filtered,  concentrated,  and  mixed  with  an  equal 
volume  of  a  '5  per  cent,  solution  of  carbolic  acid.  The  dose  is  1  c.c., 
and  it  is  used,  like  tuberculin,  for  diagnostic  purposes.  If  the 
suspected  animal  reacts  to  the  injection,  it  is  suffering  from  glanders. 
Keaction  is  judged  by  three  signs,  (a)  a  rise  of  temperature  2-3°  C., 
(b)  a  large  "  soup-plate "  swelling  at  the  site  of  inoculation,  and  (c) 
an  enlargement  of  the  lymphatic  glands. 

In  1903  there  were  in  Great  Britain  as  many  as  1463  outbreaks 
of  glanders  in  which  2490  horses  were  attacked.  This  is  the  highest 
number  of  outbreaks  since  1892,  when  they  numbered  1657.  The 
prevalence  of  the  disease  is  localised  often  to  certain  counties  and 
districts.  In  1903,  855  of  the  1463  outbreaks  occurred  in  the  county 
of  London. 


CHAPTEE  X 

TUBERCULOSIS   AS   A   TYPE  OF   BACTERIAL  DISEASE 

Pathology  and  Bacteriology  of  Tuberculosis — The  Bacillus  of  Koch — Animal  Tuber- 
culosis, Bovine,  Avian,  etc. — Bovine  and  Human  Tubercle  Bacilli  compared 
— Intercommunicability — Diagnosis  of  Bovine  Tubercle — The  Prevention  of 
Tuberculosis — Pseudo-Tuberculosis — Acid-fast  Bacteria  Allied  to  the  Tubercle 
Bacillus  :  in  Man,  in  Animals,  in  Butter  and  Milk,  in  Grass— Differential 
Diagnosis— Streptothrix  Group. 

TUBERCULOSIS  is  from  several  points  of  view  the  type  of  bacterial 
disease  which  most  concerns  the  public  health.  Its  bacteriology  is 
perhaps  more  worked  out  than  that  of  any  other  disease.  Its  pre- 
valence in  all  parts  of  the  world,  and  among  animals  as  well  as  man, 
makes  it  a  disease  of  vital  importance  and  interest  to  man.  More- 
over, the  growth  of  our  knowledge  respecting  it  has  led  to  the 
introduction  of  methods  of  prevention,  and  the  world  is  beginning 
to  understand  that  a  scientific  control  of  this  disease  is  becoming 
possible.  For  these  reasons  it  is  desirable  to  treat  somewhat  fully 
of  the  chief  facts  respecting  it. 


Pathology  and  Bacteriology  * 

As  far  back  as  1794,  Baillie  drew  attention  to  the  grey  miliary 
nodules  occurring  in  tuberculous  tissue,  which  gave  rise  to  the  term 
"tubercles."  This  observation  was  confirmed  by  Bayle  in  1810. 
In  1834  Laennec  described  all  caseous  deposits  as  "tubercles," 
insisting  upon  four  varieties : — 

*  A  detailed  study  of  tuberculosis  from  its  pathological  and  bacteriological  aspect 
will  be  found  in  La  Tuberculose  et  son  Bacille,  part  i.,  Straus,  Professor  a  la  Faculte 
de  Medecine  de  Paris ;  and  in  Tuberculosis,  by  Professor  Cornet,  edited  by  W.  B. 
James  and  A.  Stengel,  1904. 

325 


326     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

1.  Miliary,   which   were   about   the   size    of    millet   seeds,   and 
generally  occurring  in  groups. 

2.  Crude,  miliary  tubercles  in  yellow  masses. 

3.  Granular,  similar  to  the  last,  but  scattered. 

4.  Encysted,  a  hard  mass  of  crude  tubercle  with  a  fibrous  or 
semi-cartilaginous  capsule. 

The  "  tubercle "  possesses  a  special  structure,  although  it  is  not 
always  apparent,  and  certain  cell-forms  occur  in  it  and  give  it  a 
more  or  -less  characteristic  appearance. 

The  typical  lesion  is  a  nodule  of  granulation  tissue,  as  small  as 
the  size  of  a  millet  seed.  The  centre  consists  of  one  or  more 
multinucleated  cells  known  as  giant  cells,  immediately  surrounded 
by  a  zone  of  slightly  elongated  cells  with  a  somewhat  faintly-staining 
nucleus,  termed  epithelioid  cells,  owing  to  their  origin.  These  cells  in 
their  turn  are  surrounded  by  another  zone  of  small  round  cells  which 
have  but  little  cell  protoplasm,  yet  contain  a  deeply-staining  nucleus, 
and  are  known  as  lymphoid  cells.  They  are  apparently  identical 
with  lymphocytes.  The  whole  nodule  is  inflammatory  tissue  pro- 
duced as  a  result  of  the  action  of  a  specific  irritant,  namely,  the 
tubercle  bacillus. 

It  was  not  till  1865  that  the  specific  nature  of  tuberculosis  was 
asserted  by  Villemin.  Burdon  Sanderson  (1868-9)  in  England  con- 
firmed his  work,  and  it  was  extended  by  Cohnheim,  who  a  few  years 
later  laid  down  the  principle  that  all  is  tubercular  which  by  trans- 
ference to  susceptible  animals  is  capable  of  inducing  tuberculosis, 
and  nothing  is  tubercular  unless  it  possesses  this  property. 

Klebs  (1877)  and  Max  Schiller  (1880)  described  masses  of  living 
cells  or  micrococci  in  many  tuberculous  nodules  in  the  diseased 
synovial  membrane  of  joints  and  in  lupus  skin.  In  1881  Toussaint 
declared  that  he  had  cultivated  from  the  blood  of  tubercular  animals 
and  from  tubercles  an  organism  which  was  evidently  a  micrococcus, 
and  in  the  same  year  Aufrecht  stated  that  the  centre  of  a  tubercle 
contained  small  micrococci,  diplococci,  and  some  rods.  But  it  was 
not  till  the  following  year,  1882,  that  Koch  discovered  and  demon- 
strated beyond  question  the  specific  Bacillus  tuberculosis. 

It  is  now  held  to  be  absolutely  proved  that  the  introduction  of 
this  bacillus,  or  its  spores,  is  the  one  and  only  essential  agent  in  the 
production  of  tuberculosis.  Its  recognised  manifestations  in  the 
body  of  man  are  as  follows : — Tuberculosis  in  the  lungs  =  acute  or 
chronic  phthisis ;  in  the  skin  =  lupus;*  in  the  mesenteric  glands  = 
tabes  mesenterica ;  in  the  brain  =  hydrocephalus ;  in  lymphatic 
glands  =  scrofula* 

The  disease  may  occur  generally  throughout  the  body,  or  it  may 

*  There  are,  obviously,  differences  of  virulence  between  these  conditions  and 
pulmonary  tubercle. 


KOCH'S  TUBERCLE  BACILLUS  327 

occur  locally  in  the  lungs,  liver,  glands,  intestine,  larynx,  bones, 
kidneys,  spleen,  and  other  parts. 

We  may  summarise  the  history  of  the  pathology  of  tubercle 
thus : — 

1794.  Baillie  drew  attention  to  grey  miliary  nodules  occurring 
in  tuberculosis,  and  called  them  "  tubercles." 

1834.  Laennec  described  four  varieties:  miliary ;  crude;  granu- 
lar; encysted. 

1843.  Klencke  produced  tuberculosis  by  intravenous  injection 
of  tubercular  giant  cells. 

1865.  Villemin  demonstrated  infectivity  of  tubercular  matter  by 
inoculation  of  discharges;  Cohnheim,  Armanni,  Burdon 
Sanderson,  Wilson  Fox,  and  others  showed  that  nothing 
but  tubercular  matter  could  produce  tuberculosis. 

1877.  Living  cells  were  found  in  tubercles,  "  micrococci "  (Klebs, 
Toussaint,  Schiller). 

1882.  Koch  isolated  and  described  the  specific  bacillus,  and 
obtained  pure  cultivations  (1884). 

The  Bacillus  of  Koch 

Biology. — The  B.  tuberculosis  of  artificial  culture  is  usually  an 
unbranched,  slender,  immotile  rod,  1'5  to  4  /z  long  and  '4  //,  broad, 
often  slightly  bent.  In  sputum  and  tissues  the  bacillus  may  appear 
branched  and  in  thread  forms.  The  protoplasm  of  the  bacillus 
consists  of  fat  and  wax  (26  per  cent.),protamin  (24  per  cent.),  nucleo- 
proteid  (23  per  cent.),  nucleic  acid  (8  per  cent.),  and  the  remainder 
of  mineral  and  proteinoid  (chitin)  substances.  The  protoplasm  is 
frequently  vacuolated  and  irregularly  segmented,  and  this  becomes 
particularly  obvious  after  staining.  As  to  staining,  the  bacillus  is 
acid-proof,  and  stains  well  with  Ziehl-Neelsen  or  Gram.  Klein  and 
Marmorek  have  shown  that  very  young  tubercle  bacilli  are  not 
resistant  to  acid  and  alcohol.  Growth  does  not  occur  in  the  absence 
of  oxygen,  is  most  favoured  by  a  temperature  varying  from  29°  C. 
to  42°  C.,  and  is  at  all  times  slow  on  artificial  media.  In  sputum 
and  in  tissues  it  will  be  found  that  many  of  the  bacilli  are  straight 
with  rounded  ends;  others  are  slightly  curved.  They  are  usually 
solitary,  but  may  occur  in  pairs,  lying  side  by  side  or  in  small 
masses.  They  are  chiefly  found  in  fresh  tubercles,  more  sparingly 
in  older  ones.  Some  lie  within  the  giant  cells ;  others  lie  outside. 
When  stained,  they  appear  to  be  composed  of  irregular  cubical  or 
spherical  granules  within  a  faintly-stained  sheath.  In  recent  lesions 
the  protoplasm  appears  more  homogeneous,  and  only  takes  on  the 
segmented  or  beaded  character  in  old  lesions,  pus,  or  sputum.  As 
a  rule,  the  capsule  stains.  There  are  no  flagella.  So  far  as  is  known, 


328     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

the  bacillus  tuberculosis  discovered  by  Koch  is  the  only  immediate 
cause  of  all  forms  of  human  tuberculosis.  In  the  majority  of  cases 
the  micro-organism  is  met  with  in  the  form  of  slender  rods,  but  under 
certain  conditions  at  present  imperfectly  understood,  the  micro-organism 
may  show  filaments,  true  dichotomous  branching  and  club  forma- 
tion, and,  in  the  tissues,  especially  in  experimental  tuberculosis,  it  may 
assume  a  radiate  arrangement — characters  which  from  a  taxonomic 
point  of  view  bring  it  into  close  relation  with  a  large  group  of  micro- 
organisms variously  designated  Streptothricese,  Oospora,  Nocardiaceae, 
and  more  recently  Actinomycetes  (Lachner-Sandoval).  According 
to  all  experience,  the  tubercle  bacillus  is  an  aerobic  facultative 
parasite,  which  grows  extremely  slowly  outside  the  body,  and  the  fact 
that  for  its  growth  it  requires  a  relatively  high  temperature  is  against 
the  supposition  that  the  tubercle  bacillus  multiplies  extra-corporeal! y, 
at  least  in  temperate  climates. 

Morphological  differences  are  found  under  different  circumstances, 
and  within  limits  variation  occurs  according  to  the  environment. 
The  filaments,  threads,  and  true  branching  forms  of  old  cultures  have 
been  met  with,  though  only  occasionally,  in  sputum.  Clubbed 
actinomycotic  forms  have  also  been  described.  On  these  facts  some 
bacteriologists  are  disposed  to  look  upon  the  tubercle  bacillus  as 
belonging  to  the  higher  bacteria  (Plate  18). 

Cultivation  on  Various  Media. — Koch  inoculated  solid  blood  serum 
with  tubercular  matter  from  an  infected  lymphatic  gland  of  a  guinea- 
pig,  and  noticed  the  first  signs  of  growth  in  ten  or  twelve  days  in 
the  form  of  whitish,  scaly  patches.  These  enlarged  and  coalesced 
with  neighbouring  patches,  forming  white,  roughened,  irregular  masses. 
The  blood  serum  is  not  liquefied.  Nocard  and  Eoux  showed  that  by 
adding  5  to  8  per  cent,  of  glycerine  to  the  media  commonly  used  in  the 
laboratory,  such  as  nutrient  agar  or  broth,  better  growth  is  obtained. 
In  glycerine  broth  abundant  growth  appears  at  the  end  of  seven 
or  eight  days,  and  eventually  cultures  taken  from  glycerine  broth 
will  be  found  to  grow  well  in  ordinary  bouillon.  A  pellicle  generally 
forms.  On  glycerine  agar,  minute  crumb-like  colonies  of  whitish- 
yellow  colour  appear  in  six  to  twelve  days.  Later,  the  whole  growth 
turns  browner  in  colour,  and  is  sometimes  dry,  sometimes  moist,  in 
appearance  depending  on  age  of  culture,  and  consistence  of  medium. 
Ultimately,  the  discrete  colonies  coalesce  and  form  a  lichenous 
growth.  By  continuous  sub-culture  on  glycerine  agar  the  virulence 
of  the  bacillus  is  diminished.  But  in  fifteen  days  after  inoculation  of 
the  medium  the  culture  equals  in  extent  a  culture  of  several  weeks'  age 
on  blood  serum.  In  alkaline  broth  to  which  a  piece  of  boiled  white 
of  egg  was  added,  Klein  obtained  copious  growth,  and  found  that 
continued  sub-culturing  upon  this  medium  also  lessens  the  virulence. 
On  potato  the  tubercle  bacillus  grows  well  in  crumb-like  masses. 


PLATE  23. 


Bacillus  tuberculosis. 

In  sputum  from  a  case  of  human  phthisis. 

Stained  by  Ziehl-Neelsen  method. 

x   ]000. 


Bacillus  tuberculosis. 

Giant  cell.      Bacilli  in  situ  within  the  cell. 

Stained  by  Ziehl-Neelsen  method. 

x   1000. 


* 


Bacillus  tuberculosis. 

From  edge  of  caseous  patch  in  human  lung. 
x   750. 


Bacillus  tuberculosis. 

Film  preparation  from  glycerin e-glucose-agar  culture 
4  weeks  at  37°  C.      Stained  with  carbol  fuchsin    ' 
X   1000. 


[To  face  page  328. 


KOCH'S  TUBERCLE  BACILLUS  329 

Spore  formation. — In  very  old  cultivations  spore-like  bodies  can 
be  observed  both  in  stained  and  unstained  preparations,  but  neither 
the  irregular  granules  within  the  capsule  nor  the  unstained  spaces 
between  the  granules  are  spores  (Babes  and  Crookshank).  That  the 
bacilli  probably  possess  spores  is  believed  on  account  of  their 
behaviour  under  certain  circumstances.  For  example,  tubercular 
sputum  when  thoroughly  dried  retains  its  virulent  character.  Even 
cultures  of  tubercle  artificially  dried  retain  their  virulence.  Now, 
no  sporeless  bacillus  is  known  at  present  which  can  withstand  thorough 
desiccation.  Again,  non-spore-bearing  bacilli  are  killed  with  a  less 
exposure  to  heat  than  that  which  is  required  to  destroy  tubercular 
sputum.  Koch,  Lingard,  Klein,  and  others  long  ago  pointed  out  the 
resistance  of  the  bacilli  of  tubercle  to  solutions  of  perchloride  of  mercury 
and  to  heating  in  suspension  in  salt  solution,  whilst  sporeless  bacilli 
succumbed  to  the  same  treatment.  So  that  it  is  commonly  believed 
that  B.  tuberculosis  produces  spores,  even  though  such  have  not  been 
demonstrably  proved. 

Koch  and  other  bacteriologists  have  declared  the  bacillus  to  be 
a  "  true  parasite."  Koch  based  this  view  upon  the  belief  which  he 
entertained  that  the  bacillus  can  only  grow  between  30°  C.  and 
41°  C.,  and  therefore  in  temperate  zones  is  limited  to  the  animal 
body,  and  can  only  originate  in  an  animal  organism.  "  They  are," 
he  said,  "true  parasites,  which  cannot  live  without  their  hosts. 
They  pass  through  the  whole  cycle  of  their  existence  in  the  body." 
But  at  length  Koch  and  others  overcame  the  difficulties  and  grew 
the  bacillus  as  a  saprophyte.  Schottelius*  has  observed  that 
tubercle  bacilli  taken  from  the  lung  of  phthisical  persons  buried  for 
years  still  retains  its  virulence  and  capability  of  producing  tuber- 
culosis upon  inoculation.  He  further  showed  that  tubercular  lung 
kept  in  soil  (enclosed  in  a  box)  revealed  a  marked  rise  in  temperature. 
Klein  quotes  these  experiments  as  indications  that  "  tubercle  bacilli 
are  not  true  parasites,  but  belong  to  the  ectogenic  microbes  which 
can  live  and  thrive  independently  of  a  living  host." 

It  has  now  been  abundantly  proved  that  the  tubercle  bacillus 
is  capable  of  accommodating  itself  to  circumstances  much  less 
favourable  than  had  been  supposed,  as  regards  temperature  and 
environment.  For  it  is  now  known  that  it  is  possible  to  grow  the 
bacillus  upon  glycerine  agar  at  28°  C.  (82°  F.),  obtaining  an  ample 
culture  which  develops  somewhat  more  slowly  than  on  blood  serum, 
and  to  a  less  extent  than  at  37°  C.  Sheridan,  Delepine,  Czaplewski, 
Eansome,  Beevor,  and  others  have  also  been  successful  in  obtaining 
growths  at  room  temperature  both  in  summer  and  winter.  Moeller 
succeeded  in  growing  the  bacillus  at  20°  C.,  after  passing  it  through 
a  blindworm. 

*  Centralblatt.  f.  Bact,  und  Parasit, ,  vol.  vii. ,  p.  9. 


330     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

The  Relation  of  the  Bacillus  to  the  Disease.— Having  con- 
sidered the  structure  of  "  tubercles  "  and  the  chief  biological  facts  of 
the  tubercle  bacillus,  we  may  now  ask :  How  does  the  bacillus  set  up 
the  changes  in  normal  tissues  which  result  in  tubercular  nodules  ? 
In  arriving  at  a  solution  of  this  problem,  we  are  materially  aided  if 
we  bear  in  mind  the  fact  that  when  such  an  organism  is  present 
in  the  tissues  it  has  a  double  effect.  First,  there  is  an  ordinary 
inflammatory  irritation ;  and,  secondly,  there  is  a  specific  change  set 
up  by  the  toxins  of  the  bacillus.  Many  authorities  believe  that  the 
process  is,  generally  speaking,  as  follows : — Directly  the  invading 
bacilli  find  themselves  in  a  favourable  nidus  they  commence  multi- 
plication. In  the  course  of  a  few  days  this  acts  as  an  irritant  upon 
the  surrounding  connective-tissue  cells,  which  proliferate,  and  become 
changed  into  the  large  cells  known  as  epitlielioid  cells.  At  the  periphery 
of  this  collection  of  epitlielioid  cells,  we  have  a  congested  area  filled 
with  lymphocytes  drawn  thither  by  the  process  of  inflammation  and 
constituting  the  zone  of  lymphoid  cells.  The  production  of  the 
bacillary  poisons  changes  the  epithelioid  cells  in  the  centre  of  the 
nodule,  some  of  which  become  fused  together,  whilst  others  expand 
and  undergo  division  of  nucleus.  By  this  means  we  obtain  a  series 
of  large  multinucleated  cells,  giant  cells.  Thus  is  formed  the  typical 
"  tubercle."  But  if  the  disease  is  very  active,  this  soon  caseates  and 
breaks  down  in  the  centre.  In  a  limb  we  get  a  discharge;  in  a 
lung  we  get  an  expectoration.  Both  discharge  and  expectoration 
arise  from  a  breaking  down  of  the  new  cell  formation.  Previously 
to  breaking  down  we  have  in  a  fully  developed  nodule  commencing 
at  the  periphery  where  the  normal  tissue  is,  healthy  tissue,  then  the 
inflammatory  zone  of  lymphoid  cells,  then  epithelioid  cells,  and  in 
the  centre  giant  cells,  containing  nuclei  and  bacilli.  The  sputum 
or  the  discharge  will,  during  the  acute  stage  of  the  disease,  at  all 
events,  contain  countless  numbers  of  the  bacilli,  which  may  thus 
be  readily  detected,  and  their  presence  used  as  evidence  of  the 
disease.  It  is  obvious  that  if  the  centre  of  the  nodule  degenerates 
and  comes  away  as  a  purulent  discharge,  a  cavity  will  be  left  behind. 
By  degrees  this  small  cavity  becomes  enlarged,  as  is  frequently  the 
case  in  the  lung,  which  particularly  lends  itself  to  such  a  condition. 
Hence,  though  at  the  outset  the  affected  part  of  a  tubercular  lung 
becomes  solid,  ultimately  the  affected  part  becomes  a  cavity,  unless 
repair  sets  in,  and  by  growth  of  fibrous  tissue  the  commencing  cavity 
is  obliterated. 

The  exact  period  of  giant  cell  formation  depends  on  the  rapidity 
of  the  formative  inflammatory  processes.  Thus  different  conditions 
occur.  Giant  cells  are  a  constant  feature  of  interstitial  tubercles 
in  connective  tissue,  but  in  uncomplicated  caseous  tubercular 
pneumonia  there  may  not  be  found  a  single  giant  cell  in 


ACTION  OF  THE  BACILLUS  331 

a  whole  lung.  Some  authorities  look  upon  giant  cell  formation  as 
a  sign  of  chronicity  of  the  process.  Further,  in  some  of  the  lower 
animals,  the  giant  cells  become  packed  with  tubercle  bacilli,  while 
in  man  it  frequently  occurs  that  few  or  none  at  all  are  found. 
When  the  giant  cells  do  contain  bacilli  they  are  usually  arranged 
in  one  of  four  ways :  (a)  polar,  (b)  zonal,  (c)  mixed,  or  (d)  at  the 
periphery  of  the  giant  cell.  The  breaking  down  of  the  nodule  is 
partly  due  to  the  bacterial  poisons,  and  partly  to  the  nodule  being 
non-vascular,  owing  to  the  fact  that  new  capillaries  cannot  grow  into 
the  dense  nodule,  and  the  old  ones  are  occluded  by  the  growth  of 
the  nodule. 

At  first  the  disease  is  local,  owing  to  the  unfavourable  action  of 
the  blood,  to  phagocytic  action,  or  to  the  fewness  of  the  number 
of  bacilli  absorbed.  From  the  local  foci  of  disease  the  tuberculous 
process  spreads  chiefly  by  three  channels : — 

(a)  By  the  lymphatics,  affecting  particularly  the  glands.      Thus 
we  get  tuberculosis  set  up  in  the  bronchial,  tracheal,  mediastinal, 
and    mesenteric    glands,    and    so    frequently    present    as    to    be    a 
characteristic   of   the   disease.      This    is   the    common    method    of 
dissemination   in   the    body,   and   by   this   channel    the    virus    of 
tuberculosis  is  carried  along  with  the  stream  of  lymph  and  infects 
progressively  the  lymph  vessels  and  glands.     It  may  also  be  pro- 
pagated along  the  lymphatics  in  an  opposite  direction  to  the  lymph 
stream. 

(b)  By   the   blood-vessels,   by   means    of    which    bacilli    may   be 
carried    to    distant    organs.      But    this    channel    is    comparatively 
rare.       Blood     is    not    a    favourable     medium    for    the     tubercle 
bacillus. 

(e)  By  continuity  of  tissues,  that  is  by  infective  giant  cell  systems 
encroaching  upon  neighbouring  tissues,  or  discharge  from  lungs  or 
bronchial  glands  obtaining,  for  example,  entrance  to  the  gullet  and 
thus  setting  up  intestinal  disease. 

It  has  been  abundantly  proved  that  the  respiratory  and  digestive 
systems  are  those  principally  affected  by  the  tubercle  bacillus. 
Wherever  the  bacilli  are  arrested,  they  excite  formation  of  granula- 
tions or  miliary  tubercular  nodules,  which  increase  and  eventually 
coalesce.  The  lymphatic  glands  which  collect  the  lymph  from  the 
affected  region  are  earliest  affected,  always  the  nearest  first,  and  for 
a  time  the  disease  may  appear  to  be  appreciably  stopped  on  its 
invading  march.  Each  lymphatic  gland  acts  as  a  temporary  barrier 
to  progress  until  the  disease  has  broken  its  structure  down.  It 
remains  "  local,"  in  spite  of  increase  in  number  and  importance  of 
the  foci  of  disease,  as  long  as  the  bacilli  have  not  gained  access  to 
the  body  generally. 

Channels  of  Infection, — The  common  methods  of  invasion  by 


332     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

which  the  tubercle  bacillus  gains  access  to  the  human  body  are  three, 
namely,  through  the  skin,  and  through  the  alimentary  and  respiratory 
systems.  A  great  variety  of  cases  of  skin  infection  are  on  record, 
although  the  uninjured  epidermis  affords  a  fairly  reliable  protection,  so 
that  simple  contact  with  tuberculosis  sputum  does  not  suffice  to  pro- 
duce infection  if  the  skin  be  uninjured.  The  exact  means  and  occasion 
of  entry  are  innumerable.  Wounds  play  a  great  part  in  rendering 
possible  the  invasion  by  tubercle  bacilli.  Infection  by  the  alimentary 
tract  takes  place  in  a  variety  of  ways.  The  bacilli  may  be  carried 
in  with  air  in  mouth-inspiration,  by  dirty  objects  placed  in  the  mouth 
(in  children),  by  kissing  tuberculous  persons,  or  by  the  ingestion  of 
infected  food.  Thus,  we  may  have  tuberculosis  of  the  mouth  and 
tonsils,  of  the  stomach,  and  pf  the  intestine  and  other  abdominal 
organs,  including  the  mesenteric  glands.  Elsewhere  we  remark  upon 
the  comparative  rarity  of  primary  abdominal  tuberculosis  in  man, 
though  the  disease  is  more  common  in  animals. 

The  chief  channel  of  infection  is,  of  course,  the  respiratory  tract, 
and  the  two  means  by  which  tubercle  bacilli  thus  reach  the  body 
are  (a)  inhalation  of  the  dust  of  dried  tuberculous  sputum,  and 
(b)  the  inhalation  of  moist  particles  from  the  cough-spray  of  a 
phthisical  patient.  "Wherever  tuberculous  sputum  is  allowed  to  dry 
the  risks  are  great  that  the  dust  so  produced  may  be  inhaled  in  a 
virulent  form,  and  lodging  at  one  or  more  points  may  set  up  varying 
degrees  of  tuberculosis.  This  broad  fact  is  based  upon  experimental 
and  clinical  evidence.  Tuberculosis  has  been  produced  experimentally 
in  animals  in  this  way,  and  there  is  clinically  the  overwhelming- 
frequency  of  tuberculosis  of  the  lungs  among  men  exposed  to  just 
such  a  manner  of  infection.  But  Koch,  Fliigge,  and  others  have 
shown  that  not  only  is  sputum  a  source  of  infection  when  dried 
and  pulverised,  but  also  when  disseminated  by  coughing,  shouting, 
etc.,  in  the  form  of  minute  moist  particles  of  spray.  Koch  exposed 
rabbits,  guinea-pigs,  rats,  and  mice  to  an  infected  spray  for  half  an  hour 
on  three  successive  days,  and  produced  tuberculosis  in  every  animal. 
Heymann  found  that  such  spray  particles  from  human  beings 
inoculated  into  guinea-pigs  produced  tuberculosis.  Most  of  the 
droplets  are  large  and  settle  rapidly,  but  some  may  remain  suspended 
in  the  air  for  more  than  an  hour,  retaining,  of  course,  their  virulent 
properties.  Heymann  found  the  duration  of  life  of  the  bacilli  in  these 
droplets  was  eighteen  days  in  the  dark,  and  three  days  when  exposed 
to  light.  Under  ordinary  circumstances  and  an  absence  of  draughts, 
the  zone  of  danger  from  a  coughing  consumptive  extends  to  a 
distance  of  about  three  feet.  It  must  be  remembered  that  the 
tubercle  bacilli  in  the  moist  particles  of  cough-spray,  are  probably  of 
higher  virulence  than  those  in  dried  sputum  dust,  and  therefore  it 
seems  reasonable  to  suppose  that  the  cough-spray  is  the  most 


IN  BOVINES  333 

dangerous  channel  of  infection  in  tuberculosis.*  At  the  same  time 
experience  shows  that  the  degree  of  inf  ectivity  of  phthisis  is  not  a  very 
high  one.  It  is  a  truly  infective  disease,  but  not  an  extremely 
infectious  disease.  It  may  be  rightly  described  as  sub-infectious.\ 

Toxins  of  the  Tubercle  Bacillus. — Many  investigators  have  isolated 
products  from  pure  cultures  of  the  tubercle  bacillus.  These  have 
comprised  chiefly  albumoses,  alkaloids,  various  extractives,  and  inorganic 
salts.  Koch  isolated  "  tuberculin  "  from  cultures  of  tubercle  bacillus 
upon  glycerine  broth  by  means  of  evaporation  and  precipitation  with 
alcohol.  Buchner  obtained  by  trituration  and  compression  of  fresh 
tubercle  bacilli  a  substance  termed  "  tuberculo-plasmine."  But  of 
the  real  nature  of  the  toxins  of  the  tubercle  bacillus  little  is  known. 


Bovine  Tuberculosis 

Cattle  come  first  amongst  animals  liable  to  tubercle.  Horses  may 
be  infected,  but  it  is  comparatively  rare,  and  among  small  ruminants 
the  disease  is  rarer  still.  Dogs,  cats,  and  kittens  may  be  easily 
infected.  Amongst  birds,  fowls,  pigeons,  turkeys  and  pheasants 
the  disease  assumes  almost  an  epidemic  character.  Especially  do 
animals  in  confinement  die  of  tubercle,  as  is  illustrated  in  zoological 
gardens. 

Bovine  Tuberculosis. — Respecting  the  lesions  of  bovine  tuberculosis, 
it  will  be  sufficient  to  say  that  nothing  is  more  variable  than  the 
localisation  or  form  of  its  attacks.  The  lungs  and  lymphatic  glands 
come  first  in  order  of  frequency,  next  the  serous  membranes,  then 
the  liver  and  intestine,  and  lastly  the  spleen,  joints,  and  udder 
(Nocard).  The  anatomical  changes  in  bovine  tubercle  are  mostly 
found  in  the  lungs  and  their  membranes,  the  pleurse.  It  also  affects 
the  abdomen  and  its  chief  organs,  the  peritoneum,  and  the  lymphatic 
glands.  In  both  of  these  localities  a  characteristic  condition  is  set 
up  by  small  grey  nodules  appearing  on  the  pleura  and  peritoneum, 
the  nodules,  increasing  in  size,  giving  an  appearance  of  "grapes." 
Hence  the  condition  is  called  grape  disease,  or  Perlsuckt.  The  organs, 
as  we  have  said,  are  equally  affected,  and  when  we  add  the  lymphatic 
glands  we  have  a  fairly  complete  summary  of  the  form  of  the  disease 
as  it  occurs  in  cattle.  In  about  half  of  all  cases  the  lungs  and  serous 
membranes  become  simultaneously  affected,  in  about  one-third  the 
lungs  alone;  and  in  about  one-fifth  the  serous  membranes  alone 
(Friedberger  and  Frohner).  As  has  been  pointed  out  by  Martin, 

*  For  a  discussion  on  the  channels  of  infection  in  tuberculosis,  see  Garnet, 
Tuberculosis,  1904,  pp.  96-282;  Fliigge,  Zeitschrift  fur  Hyg.  u.  Jufek.,  Band 
xxxviii.,  1901. 

t  Koch,  Etiology  of  Tuberculosis  ;  in  Brit.  Med.  Jour.,  1903,  i.,  p.  593  (Hillier), 
will  be  found  a  useful  summary  of  modern  views  on  the  question. 


334    TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

Woodhead,  and  others  in  their  evidence  before  the  Eoyal  Commis- 
sion, the  organs,  glands,  and  membranes  are  the  common  sites  for 
tubercle,  not  the  muscles  (or  meat). 

The  following  table  records  the  findings  of  Geddes,  who  in  1901- 
1902  was  sent  by  the  American  Government  to  examine  by  means 
of  tuberculin  some  of  the  chief  breeds  of  British  dairy  cattle.* 


Breed. 

No.  Tested. 

No.  Rejected. 

Percentage 
of  Rejections. 

Jersey  (in  Great  Britain) 

42 

23 

54-76 

Aberdeen  —  Angus 

258 

104 

28-73 

Ayrshire     . 

33 

8 

24-24 

Shorthorn  . 

228 

53 

23-25 

Guernsey  (in  Great  Britain) 

57 

11 

19-30 

Galloway    . 

36 

6 

16-67 

Highland   . 

19 

3 

15-79 

Red  Polled 

57 

4 

7-02 

Hereford    . 

428 

17 

3-97 

Jersey  (on  island) 

324 

1 

0-31 

Dexter  Kerry     . 

15 

0 

o-oo 

Guernsey  (on  island) 

53 

0 

o-oo 

Sussex 

1 

0 

o-oo 

Total. 

1551 

230 

14-77 

Eliminating  the  tests  on  Guernsey  and  Jersey,  the  proportions  of 
reactions  among  the  tests  made  in  Great  Britain  and  Ireland  were  as 
follow :— in  1901, 13-67  per  cent. ;  in  1902,  20'97 ;  and  for  both  years, 
17'92.  Hopkins  examined  571  Shorthorns  and  found  the  percentage 
of  positive  reaction  was  23'0  as  compared  with  Geddes's  result  of 
23'25.f 

Tuberculosis  of  the  udder  is  comparatively  rare.  Out  of  100 
tuberculous  cows  not  more  than  3  or  4  have  tuberculosis  of  the 
udder  (Bang).  The  disease  occurs  as  a  diffuse,  slightly  hard,  enlarge- 
ment, generally  unaccompanied  by  fever  or  tenderness  of  the  organ. 
Usually  only  one  quarter  is  attacked,  and  that  generally  a  posterior 
quarter.  The  gland  lobules  become  hypertrophied,  and  the  larger 
milk-ducts  contain  yellowish  caseous  masses  full  of  bacilli.  As  the 
condition  advances,  there  is  a  considerable  increase  of  the  inter- 
lobular  connective  tissue  (interstitial  mastitis)  of  the  nature  of  a 
sclerosis,  and  firm  tubercles  of  various  sizes  begin  to  appear.  Con- 
sequent upon  these  changes  the  udder  becomes  nodular,  and  hard 

*  Nineteenth  Annual  Report  of  the  Bureau  of  Animal  Industry,  1902,  p.  551. 
|  Report  of  Minister  of  Agriculture,  Dominion  of  Canada,  1902,  p.  134. 


TUBERCULOUS  MILK  335 

and  tough.  Miliary  tubercles  appear  in  the  walls  of  acini,  and 
enormous  deposits  of  bacilli  may  be  found  in  the  udder.  Simultane- 
ously with  these  changes,  the  mammary  lymphatic  glands  (pudic 
glands)  lying  above  the  posterior  region  of  the  udder  became 
enlarged,  indurated,  and  caseous.  The  disease  may  advance  slowly  or 
with  great  rapidity.  But  finally  the  condition  is  such  that  the 
glandular  tissue  of  the  udder  is,  as  it  were,  smothered  by  the  hyper- 
trophy and  fibrous  transformation  of  the  interstitial  connective  tissue. 
The  large  excretory  ducts  become  blocked  by  granulations  or  fibrous 
growth  outside  them,  or  by  caseous  masses  inside.  This  stage 
inevitably  leads  to  milk  suppression  (see  also  p.  203). 

It  should  not  be  forgotten  that  tuberculosis  of  the  udder  is 
associated  with  tuberculosis  of  the  internal  organs.  It  is  almost 
invariably  secondary.  It  may  exist  with  mild  or  advanced  disease 
of  the  internal  organs.  Its  diagnosis  is  all  the  more  difficult 
owing  to  the  fact  that  there  may  be  no  symptoms.  Generally, 
opinion  must  be  guided  by  the  local  condition  of  the  udder,  coupled 
with  the  condition  of  the  milk.  It  may  occur  as  a  slow,  painless 
growth  only  evident  when  advanced,  or  it  may  increase  with  extra- 
ordinary rapidity.  This  latter  fact  makes  it  desirable  that  every 
animal  suffering  from  tuberculosis  of  however  mild  a  character  should 
be  strictly  eliminated  from  dairy  stock.  The  three  points  usually 
emphasised  for  diagnosis  of  tuberculous  udder  disease  are — (a) 
abnormal  milk  from  one  quarter,  generally  a  posterior  quarter ;  (b) 
some  hardness,  toughness,  or  irregularity  of  the  udder;  and  (c) 
enlargement  of  supra-mammary  glands.*  The  best  diagnostic  of 
general  tuberculosis  is  the  tuberculin  reaction. 

Changes  in  Milk  from  a  Tuberculous  Udder. — One  of  the  first 
signs  of  abnormality  is  the  diminution  in  the  yield.  Previously  to 
this  it  is  said  there  is  an  actual  increase  in  the  quantity  of  milk. 
As  soon  as  the  disease  begins  to  have  effect,  there  is  a  definite  decline 
in  the  yield.  For  example,  a  cow  which  in  health  gave,  say,  fifteen 
litres  of  milk,  falls  to  one  half  or  one  quarter  of  that  amount.  The 
milk  also  changes  in  consistence,  becoming  thin,  watery,  and  serous. 
At  the  same  time  the  colour  may  turn  to  yellow,  and  the  flocculi  and 
Hakes  which  occur  in  milk  from  a  healthy  udder  are  present  in 
larger  size.  As  the  yield  diminishes,  the  consistence  of  the  fluid 
becomes  more  and  more  irregular,  the  flocculi  predominating.  If 
such  milk  be  allowed  to  stand  in  a  vessel,  a  deposit  of  solid  matter, 
composed  of  these  fragments,  settles  down,  leaving  a  superficial  layer 
of  thin  fluid  at  the  top.  Finally,  the  consistence  becomes  sero- 
purulent  and  then  purulent.  Hence,  previously  to  suppression  we 
get  a  thick  yellow  purulent  fluid,  having  an  alkaline  reaction, 
coagulated  casein,  and  diminution  of  lactose.  As  a  rule,  tubercle 

*  See  also  Report  of  Royal  Commission  on  Tuberculosis,  1896,  part  iii.,  pp.  41,  42. 


336   TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

bacilli  are  readily  found,  and  whether  that  be  so  or  not  the  milk  is 
highly  infective.* 

The  Entrance  of  the  Bacillus  into  Milk. — There  are  two  main 
sources  of  the  tubercle  bacilli  found  in  milk,  namely,  a  bovine  source 
and  a  human  source.  The  two  common  channels  respectively  are  a 
tuberculous  udder  and  a  phthisical  lung.  From  the  former,  milk 
may  derive  a  direct  and  abundant  supply  of  tubercle  bacilli ;  from 
the  latter,  milk  may  become  indirectly  contaminated  by  the  parti- 
culate  matter  of  dried  sputum. 

Tuberculosis  may  be  introduced  into  healthy  cows  in  a  variety  of 
ways.  The  most  common  method  is  by  means  of  a  tuberculous 
animal,  from  the  excretions  and  discharges  of  which  infection  may 
be  conveyed  to  soil,  water,  air,  fodder,  and  general  surroundings.  In 
this  way  not  only  other  animals  cohabiting  with  a  tuberculous 
animal  become  infected,  but  premises,  stables,  and  utensils  may  also 
become  infected.  The  milk  of  a  tuberculous  animal  may  also  be 
consumed  by  other  animals  on  the  farm,  and  so  a  vicious  circle  of 
infection  is  completed.  Eavenel  has  shown  that  by  the  cough  of  as 
tuberculous  cow  tubercle  bacilli  may  be  distributed.  Of  thirty-four 
examinations  carried  out  on  five  tuberculous  cows,  tubercle  bacilli 
were  detected  on  twenty  occasions.  One  of  the  cows  constantly 
coughed  up  a  tenacious  mucus  containing  large  numbers  of  tubercle 
bacillLf  The  saliva  as  well  as  the  bronchial  mucus  of  tuberculous 
cows  has  been  found  to  contain  abundant  bacilli,  and  by  licking  her 
udder  it  is  possible  for  a  tuberculous  cow  to  convey  tubercle  bacilli 
to  its  exterior  surface. 

The  excreta  also  are  infective  when  lesions  are  located  in  the 
alimentary  canal.  In  tuberculosis  affecting  the  alimentary  canal  of 
the  cow  (1  per  cent,  of  the  cases),  it  is  thus  possible  to  get  contam- 
ination of  the  milk,  indirectly,  from  the  excreta.  The  mucous 
membrane  of  the  intestine,  especially  the  colon,  sometimes  shows 
tubercular  ulcers,  which  are  less  frequently  observed  in  the  abomasum. 
Tubercles  may  also  develop  under  the  mucous  membrane,  and  serosa 
of  the  stomach  and  intestines.  In  these  ways  arises  a  condition  of 
intestinal  tuberculosis,  which  in  its  acute  or  ulcerating  stage  will 
cause  the  excreta  to  be  loaded  with  tubercle  bacilli.  Any  one 
familiar  with  a  cowshed  will  at  once  recognise  how  readily  milk 
might  become  infected  under  such  circumstances,  which,  though 
undoubtedly  exceptional,  must  not  be  overlooked.^  In  these  ways 
stalls  may  become  infected  and  transmit  the  disease  to  fresh  herds 
stabled  in  such  premises.  Nor  are  herds  unstabled  always  free  from 

*  See  also  Report  of  Royal  Commission  on  Tuberculosis,  1896,  part  iii.,  p.  142. 
t  Commonwealth  of  Pennsylvania,  Bulletin  75  (Pearson  and  Ravenel),  1901,  p.  82. 
t  Trans.  British  Congress  on  Tuberculosis,   1901,   vol.   iii.,   p.  664  (Boinet  and 
Heron). 


BOVINE  AND  HUMAN  BACILLI  337 

tuberculosis,  as  has  been  recently  stated.  A  number  of  observers 
have  shown  that  whilst  it  is  true  that  ill-ventilated,  dark,  damp  cow- 
sheds predispose  to  infection,  milch  cows  living  entirely  in  the  open 
do  not,  on  that  account,  escape  the  disease.*  It  depends  upon  infec- 
tion in  the  herd,  that  is,  upon  contagion.  But  it  is  probable  that, 
through  more  than  any  other  channel,  the  udder  is  the  most  common 
one  for  the  conveyance  of  infection.  When  the  udder  is  affected,  the 
milk  invariably  contains  large  numbers  of  bacilli,  and  it  will  be 
understood  when  one  cow  in  a  herd  is  so  diseased,  the  entire  volume 
of  mixed  milk  from  the  herd  may  be  contaminated.  The  presence  of 
the  bacilli  in  the  milk  is  not  always  proportionate  to  the  extent  of  the 
disease  in  the  animal,  especially  when  diagnosed  clinically.  The 
reason  of  this  is  the  difficulty  of  clinical  diagnosis  between  chronic 
interstitial  mastitis  and  tuberculous  udder.  There  can,  however,  be 
little  doubt  that  the  chief  source  of  tubercle  bacilli  in  milk  is  the 
tuberculous  udder. 

Finally,  milkers  affected  with  phthisis  may  readily  infect  the 
milk,  either  by  the  repulsive  habit  of  spitting  on  their  hands  prior  to 
milking,  or  by  dried  expectoration  in  cowshed,  dairy,  or  milk-shop. 
After  distribution,  milk  is  exposed  in  a  variety  of  ways  to  dust,  and 
it  cannot  be  doubted  that  such  dust  does  at  times  contain  particulate 
matter  derived  from  dried  tubercular  expectoration,  and  that  there- 
fore in  this  way  also  it  is  possible  for  milk  to  become  infected. 

The  Bovine  and  Human  Tubercle  Bacillus  Compared.— The 
morphology  of  the  bacilli  in  cultures  of  bovine  origin  is  more 
uniform  and  constant  than  in  cultures  from  man.  The  bovine  bacilli 
are  thick,  straight,  and  short,  seldom  more  than  2  ^  in  length,  and 
averaging  less  (Theobald  Smith).  In  the  early  generations  many 
individuals  are  seen  which  are  oval,  their  length  not  more  than 
double  their  breadth.  They  are  less  granular  than  those  from  a 
human  source.  They  stain  evenly  and  deeply  with  carbol-fuchsin, 
beading  being  almost  always  absent  from  young  cultures,  and  often 
from  old  ones.  In  culture  they  have  fairly  constant  and  persistent 
peculiarities  of  growth  and  morphology  (Eavenel). 

The  human  bacilli  are,  on  the  other  hand,  much  longer,  thinner, 
and  tend  to  increase  in  length  in  sub-cultures.  They  are  generally 
more  or  less  curved,  sometimes  showing  S-shaped  forms.  They 
stain  less  intensely  with  carbol-fuchsin,  but  beading  is  generally 
seen,  even  in  early  growths,  and  is  often  very  well  marked. 

The  above  characteristics  are  most  evident  and  persistent  in 
cultures  grown  on  blood  serum.  On  glycerine  agar,  glycerine 
bouillon,  and  glycerine  potato,  bovine  and  human  tubercle  bacilli 
approach  each  other  in  cultural  features  and  morphology  much  more 
closely,  and  by  continued  cultivation  the  differences  tend  to  become 

*  Report  on  Bovine  Tuberculosis,  Government  of  New  Zealand,  1900  (Gilruth). 

Y 


338     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

obliterated.  Bovine  cultures  are  more  difficult  to  isolate  than  human, 
are  apt  to  grow  as  discrete  colonies  in  the  first  culture,  and  for 
several  generations  grow  in  a  thin  layer  which  somewhat  resembles 
ground  glass.  The  optimum  temperature  and  the  thermal  death- 
point  are  practically  the  same  in  both  forms. 

The  human  bacillus,  as  a  rule,  grows  somewhat  more  easily  and 
abundantly  from  the  first,  and  will  grow  well  on  glycerine  agar  in 
sub-cultures  made  directly  from  the  original  growth  on  blood  serum. 
All  attempts  to  obtain  a  like  result  with  the  bovine  organisms  have 
failed.  In  artificial  culture  the  human  bacillus  rapidly  loses  viru- 
lence. The  bovine  bacillus  grows  as  a  film  on  blood  serum,  whereas 
the  human  bacillus  produces  warty  growths. 

The  morphological  distinctions  tend  to  disappear  also  in  the 
tissues  of  susceptible  animals.  We  may  inoculate  a  typical  bovine 
culture,  and  in  a  short  time  obtain  from  the  various  organs  long  and 
much  beaded  bacilli  simulating  the  human  variety  (Hueppe). 

The  most  striking  dissimilarity  is,  however,  seen  in  the  action  of 
the  bacilli  from  the  two  sources  on  animals.  By  whatever  method 
of  inoculation,  the  bovine  bacillus,  as  a  rule,  possesses  a  much 
greater  pathogenic  power  than  the  human  bacillus  for  all  animals  on 
which  it  has  been  tried  (Villemin,  Eavenel,  and  others),  the  only 
exceptions  being  possibly  those  animals,  like  guinea-pigs,  which  are 
so  extremely  susceptible  to  both  types  that  it  is  difficult  to  draw  very 
much  distinction  between  them.  Dorset  and  other  workers  hold 
that  in  bovine  and  human  tuberculosis  we  have  to  do  with  organisms 
differing  usually  in  virulence,  but  between  which  there  is  no  other 
essential  distinction.* 

Intercommunicability  of  Human  and  Bovine  Tuberculosis 

Since  the  discovery  by  Koch  in  1882  of  the  tubercle  bacillus,  it 
has  generally  been  held  that  tuberculosis  in  man  and  animals  is  one 
and  the  same  disease,  f  Villemin  (1865)  was  the  first  to  main- 
tain this  identity  on  the  results  of  inoculation  of  bovine  and 
human  tubercular  matter  into  small  animals.  Chauveau  (1868) 
carried  out  similar  experiments  upon  cattle.J  Both  workers 
were  successful  in  transmitting  the  disease,  which  produced  similar 
effects  in  the  inoculated  animals.  Many  other  workers  have 
obtained  like  results,  which  were  more  or  less  uniformly  in  support 
of  the  view  that  the  identity  of  bovine  and  human  tuberculosis  was 

*  Trans.  British  Congress  on  Tuberculosis,  1901,  vol.  iii.,  pp.  553-81.  See  also 
experiment  of  Kossel  and  others,  to  which  reference  is  made  on  p.  344,  and 
U.S.  Dep.  of  Agriculture,  1904,  Bull.  52  (Dorset). 

t  Kruse,  Pansini,  Fischel,  Johne,  etc.  See  also  Twelfth  and  Thirteenth  Annual 
Reports  of  the  Bureau  of  Animal  Industry,  Washington,  1895-96  (Theobald  Smith). 

£  Congrespour  Vttude  de  la  Tuberculose,  Paris,  1888. 


INTERCOMMUNICABILITY  339 

a  thing  to  be  accepted  as  a  proved  and  fundamental  proposition. 
Not  only  have  various  workers  separately  arrived  at  that  conclusion, 
but  the  conclusions  of  the  Koyal  Commission  on  Tuberculosis,  1895, 
included  the  following  words  : — "  We  find  the  present  to  be  a  con- 
venient occasion  for  stating  explicitly  that  we  regard  the  disease  as 
being  the  same  disease  in  man  and  the  food  animals,  no  matter  though 
there  are  differences  in  the  one  and  the  other  in  their  manifesta- 
tions of  the  disease ;  and  that  we  consider  the  bacilli  of  tubercle  to 
form  an  integral  part  of  the  disease  in  each,  and  (whatever  be  its 
origin)  to  be  transmissible  from  man  to  animals,  and  from  animals 
to  animals.  Of  such  transmission  there  exists  a  quantity  of 
evidence,  altogether  conclusive,  derived  from  experiment."  * 

Whilst  there  was  up  to  1901  almost  entire  unanimity  of  opinion 
amongst  various  workers  in  respect  to  this  identity,  it  should  not  be 
supposed  that  there  was  unanimity  in  respect  to  the  degree  of 
pathogenicity.  It  was,  in  fact,  conceded  on  all  hands  that 
tuberculosis  was  a  more  virulent  disease  in  animals  than  in  man, 
and  that  the  bacillus  in  the  two  species  differed  in  various  respects 
as  to  morphological,  biological,  and  pathological  properties  (Theobald 
Smith,  Dinwiddie,  Frothingham).  In  1901,  however,  Dr  Koch 
expressed  the  opinion  that,  "  human  tuberculosis  differs  from  bovine, 
and  cannot  be  transmitted  to  cattle,"  f  and  that  bovine  tuberculosis 
was  scarcely,  if  at  all,  transmissible  to  man.  On  the  same  occasion 
counter-evidence  was  produced  by  MacFadyean,J  Ravenel,§  Crook- 
shank,  ||  and  many  others. 

As  a  result  of  experiment,  Koch  felt  "justified  in  maintaining  that  human 
tuberculosis  differs  from  bovine,  and  cannot  be  transmitted  to  cattle."  He  further 
concluded  that  bovine  tuberculosis  was  scarcely,  if  at  all,  transmissible  to  man.  It 
will  be  at  once  obvious  that  these  two  conclusions,  that  human  tuberculosis  is  not 
transmissible  to  cattle,  and  that  bovine  tuberculosis  is  not  transmissible  to  man,  are 
of  profound  and  far-reaching  importance.  Now  if  it  were  found  on  further 
investigation  that  these  conclusions  were  correct,  the  prevention  of  human 
tuberculosis  would  be  greatly  simplified,  and  the  precautionary  measures  hitherto 
adopted  for  protecting  human  food  from  infection  with  animal  tuberculosis  need 
not  be  enforced  with  the  same  stringency  as  at  present,  or,  at  least,  would  require 
considerable  modification. IT 

*  Report  of  Royal  Commission  on  Tuberculosis,  1895,  part  i.,  p.  10,  par.  23. 

t  Trans.  Brit.  Cong,  on  Tuberculosis,  1901,  vol.  i.,  p.  29. 

J  Ibid.,  vol.  i. ,  p.  79. 

§  Ibid.,  vol.  i.,  p.  91,  and  vol.  iii.,  p.  553.  ||  Ibid.,  vol.  i.,  p.  92. 

If  It  would  not  necessarily  be  justifiable  to  say  that  in  this  event  such  pre- 
cautionary measures  might  be  "  altogether. withdrawn,"  as  has  been  suggested,  for 
it  will  be  understood  that  tuberculous  meat  and  milk  from  animals  might  still  be 
unwholesome  and  unfit  for  the  food  of  man,  even  though  there  was  evidence 
to  show  that  the  exact  specific  disease  was  incommunicable.  Presumption  would 
always  be  against  the  consumption  of  meat  or  milk  plus  disease  products,  whether 
tubercle  bacilli  or  not,  for  such  food  is  not  of  the  quality  and  nature  reasonably 
expected  by  the  purchaser.  Various  non-specific  diseases  of  animals  cause  meat  to 
be  unfit  for  the  food  of  man. 


340    TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

The  evidence  furnished  by  Dr  Koch  for  the  conclusion  that  human  tuberculosis  is 
not  communicable  to  animals  is  briefly  this  :—  Nineteen  young  cattle  which  had 
stood  the  tuberculin  test  (and  were  therefore  presumably  free  from  tuberculosis) 
were  treated  as  follows :— Six  were  fed  with  tubercular  human  sputum  almost  daily 
for  seven  or  eight  months.  Four  repeatedly  inhaled  great  quantities  of  bacilli 
which  were  distributed  in  water  and  scattered  with  it  in  the  form  of  spray.  The 
remainder  (9)  were  infected  in  various  ways  with  pure  cultures  of  tubercle  bacilli 
taken  from  human  tuberculosis,  or  tubercular  sputum  direct  from  consumptive 
patients.  In  some  cases  the  bacilli  or  sputum  were  injected  under  the  skin,  in 
others  into  the  peritoneal  cavity,  and  in  others  into  the  jugular  vein.  None  of 
these  19  cattle  showed  any  symptoms  of  disease.  After  six  to  eight  months  they 
were  killed,  and  in  their  internal  organs  not  a  trace  of  tuberculosis  was  found.  The 
result  was  utterly  different,  however,  when  the  same  experiment  was  made  on 
cattle  free  from  tuberculosis  with  tubercle  bacilli  from  bovine  sources.  In  this 
case  virulent  tuberculosis  rapidly  supervened.  Further,  an  almost  equally  striking 
distinction  between  human  and  bovine  tuberculosis  was  brought  to  light  by  a 
feeding  experiment  with  swine.  Six  young  swine  were  fed  daily  for  three  months 
with  the  tubercular  sputum  of  consumptive  patients.  Six  other  swine  received 
bacilli  of  bovine  tuberculosis  with  their  food  daily  for  the  same  period.  The 
animals  that  were  fed  with  sputum  remained  healthy  and  grew  lustily,  whereas 
those  that  were  fed  with  the  bacilli  of  bovine  tuberculosis  soon  became  sickly,  were 
stunted  in  their  growth,  and  half  of  them  died.  After  three  months  and  a  half  the 
surviving  swine  were  all  killed  and  examined.  Among  the  animals  that  had  been 
fed  with  sputum  no  trace  of  tuberculosis  was  found,  except  here  and  there  little 
nodules  in  the  lymphatic  glands  of  the  neck,  and  in  one  case  a  few  gray  nodules  in 
the  lungs.  The  animals,  on  the  other  hand,  which  had  eaten  bacilli  of  bovine 
tuberculosis  had,  without  exception  (just  as  in  the  cattle  experiment),  severe 
tubercular  diseases,  especially  tubercular  infiltration  of  the  greatly  enlarged 
lymphatic  glands  of  the  neck  and  of  the  mesenteric  glands,  and  also  extensive 
tuberculosis  of  the  lungs  and  the  spleen.  The  difference  between  human  and 
bovine  tuberculosis  appeared  not  less  strikingly  in  a  similar  experiment  with  asses, 
sheep,  and  goats,  into  whose  vascular  systems  the  two  kinds  of  tubercle  bacilli 
were  injected.  Dr  Koch  also  stated  that  other  experiments  in  former  times,  and 
recently  in  America,  have  led  to  the  same  result. 

In  support  of  his  second  contention,  namely,  that  bovine  tuberculosis  is  not  trans- 
missible to  man,  Dr  Koch  points  out  that  the  direct  experiment  upon  human  beings 
is,  of  course,  out  of  the  question,  and  hence  it  is  necessary  to  rely  upon  indirect 
evidence.  Dr  Koch,  therefore,  reasons  as  follows  :  Tuberculosis,  caused  by  meat 
or  milk,  can  be  assumed  with  certainty  only  when  the  intestine  suffers  first,  i.e., 
when  a  so-called  "primary  tuberculosis"  of  the  intestine  is  found.  If  bovine 
tubercle  bacilli  are  capable  of  causing  disease  in  man  there  are  abundant  oppor- 
tunities for  the  transference  of  the  bacilli  from  one  species  to  the  other,  and  cases 
of  primary  intestinal  tuberculosis  from  consumption  of  tuberculous  milk  ought 
therefore  to  be  of  common  occurrence.  "  But  such  cases,"  he  maintains,  "  are 
extremely  rare."  In  support  of  this  view  Dr  Koch  stated  that  he  had  only  seen 
2  cases ;  that  only  10  cases  had  been  met  with  in  the  Charite  Hospital  in 
Berlin  ;  and  that  out  of  3104  post-mortems  of  tubercular  children,  Biedert  observed 
only  16  cases.  Reference  was  also  made  to  other  similar  evidence. 

Finally,  Dr  Koch  maintained  that  **  though  the  important  question  whether  man 
is  susceptible  to  bovine  tuberculosis  at  all  is  not  yet  absolutely  decided,  and  will  not 
admit  of  absolute  decision  to-day  or  to-morrow,'  one  is,  nevertheless,  already  at 
liberty  to  say  that  if  such  a  susceptibility  really  exists  the  infection  of  human  beings 
is  but  a  very  rare  occurrence. " 

Such,  then,  was  the  position  of  the  question  at  the  end  of  1901.  It  may  be  con- 
venient here  to  add  the  chief  reasons  for  supposing  that  bovine  and  human 
tuberculosis  are  one  and  the  same  disease,  and  intercommunicable : — 

1.  That  the  tubercle  bacillus  of  bovine  tuberculosis  possesses  characteristics  of 
shape,  size,  staining,  and  cultivation  on  artificial  media  similar  to,  and  in  the 
opinion  of  many  authorities  almost  identical  with,  the  tubercle  bacillus  of  human 
origin. 


INTERCOMMUNICABILITY  341 

2.  That  in  specially  prepared  and  suitable  media  artificial  cultures  of  the  tubercle 
bacillus  from  bovine  and  human  sources  have  produced  indistinguishable  effects 
when  they  have  been  employed  to  infect  a  variety  of  animals,  which  would  seem 
to  indicate  that  the  conditions  produced  are  only  variations  of  one  and  the  same 
disease. 

3.  That  tuberculin  *  produces  a  specific  reaction  in  tuberculous  cattle,  whether 
human  or  bovine  tubercle  bacilli  have  been  employed  in  its  preparation. — (Mac- 
Fadyean. ) 

[It  will  be  seen  that  these  three  reasons  have  relation  to  the  theory  of  the  identity 
of  bovine  and  human  tuberculosis.  ] 

4.  That  because  the  tubercle  bacillus  derived  from  bovine  sources  is,  either  by 
inoculation  or  ingestion  as  food,  admittedly  very  virulent  and  dangerous  for  such 
diverse  species  of  animals  as  the  rabbit,  horse,  dog,  pig,  sheep,  and  cow,  it  is 
highly  probable  that  it  is  also  dangerous  to  man.f     For  it  is  well  known  that  the 
majority  of  disease-producing  bacteria  are  harmful  to  only  one  or  two  species  of 
animals,  but  those  disease-producing  bacteria  that  are  common  to  all  the  domesticated 
animals  are  also  able  to  produce  disease  in  man. 

5.  That  the  statistics  and  percentages   set  forth  by  Dr  Koch  with  regard   to 
primary  intestinal    tuberculosis    cannot    be    accepted    as    representing    universal 
experience.     For  example,  in  two  separate  reports  from  two  children's  hospitals  in 
London  and  Edinburgh  dealing  with  547  cases  of  death  from  tuberculosis  in  children, 
it  appears  that  29  -1  per  cent,  and  28  •!  per  cent,  of  the  cases  respectively  primary 
infection  appeared  to  have  taken  place  through  the  intestine.     But  quite  apart  from 
statistics,  the  whole  question   of  such  primary  intestinal  tuberculosis  (which   Dr 
Koch  held  as  the  only  acceptable  evidence  of  tuberculous  infection  through  milk 
and  meat)  is  fraught  with  many  difficulties  and  fallacies,  and  is  at  present  sub  judice. 
It  has  been  shown  by  Professor  Sidney  Martin  and  others  that  primary  intestinal 
tuberculosis  may  not  be,  by  any  means,  an  invariable  criterion  of  tubercular  infection 
by  means  of  food  (vide  infra}. 

6.  That  there  are  on  record  a  number  of  cases  in  which  there  appeared  to  be 
substantial  evidence  to  show  that  persons  had  contracted  tuberculosis,  directly  or 
indirectly,  by  means  of  milk  or  meat.     It  is  obvious  that  such  cases,  unless  occurring 
with  extraordinary  frequency,  are  only  of  relative  value.     Moreover,  there  are  other 
channels  of  infection  to  eliminate,  and  this  it  is  often  impossible  to  do. 

7.  That  the  results  obtained  from  the  inoculation  of  human  tubercle  into  animals 
by  Dr  Koch  cannot  be  accepted  as  in  complete  accord  with  universal  experience. 
In  England  alone  somewhat  similar  experiments  have  been  performed,  having  positive 
results.     Several  years  ago  Professor  Crookshank  carried  out  such  an  experiment. 
He  obtained  sputum  containing  numerous  tubercle  bacilli  from  an  advanced  case  of 

*  Tuberculin  is  a  product  of  the  artificial  cultivation  of  the  tubercle  bacillus  (human 
or  bovine)  now  used  as  a  diagnostic  injection  test  into  cattle.  If  such  cattle  are 
suffering  from  tuberculosis  they  "  react "  (giving  high  temperature,  swelling  at  the 
point  of  inoculation,  etc.) ;  if  not  so  suffering,  they  do  not  react. 

f  See  the  researches  of  Villemin  (1865),  Klebs,  Chauveau(1867),  Gerlach,  Gunther 
and  Harms  (1870-1873),  Bellinger,  and  others.  Further,  Friedberger  and  Frohner 
state  in  their  Veterinary  Pathology  that  Wesener  compiled  reports  up  to  1884  of  369 
feeding  experiments,  the  positive  and  negative  results  of  which  were  about  equal  in 
number.  From  this  compilation  it  appears  that  (a)  71  animals,  among  which  guinea- 
pigs  and  swine  proved  most  susceptible,  were  experimented  upon  with  human  tuber- 
cular matter ;  (6)  180  experiments  were  made  with  tubercular  matter  from  cattle ; 
(c)  the  flesh  of  tuberculous  cattle  was  given  on  32  occasions  as  food,  with  the  result 
that  pigs  were  found  to  be  more  susceptible  than  other  animals,  and  that  dogs  were 
unaffected ;  and  (d)  the  milk  of  tuberculous  cows  was  given  as  food  in  86  cases. 
From  these  experiments  it  was  found  that  in  the  scale  of  comparative  racial  sus- 
ceptibility the  herbivora  (cattle,  sheep,  goats)  proved  highest,  then  swine,  and  after 
these  guinea-pigs  and  rabbits.  Carnivorous  animals  were  little  affected.  Bovine 
tubercular  matter  was  found  to  possess  the  greatest  power  of  infection,  then  came 
the  sputum  of  tuberculous  men,  then  the  milk  of  tuberculous  animals,  and  lastly, 
tuberculous  flesh. 


342     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

human  consumption.  This  was  injected  into  the  peritoneal  cavity  of  a  healthy  calf. 
The  animal  became  ill  and  died  forty-two  days  after  inoculation  from  pycemia  (blood- 
poisoning).  On  post-mortem  examination  it  was  found  that  there  were  abundant 
signs  of  generalised  tuberculosis.*  This  calf  was  not  tested  with  tuberculin  pre- 
viously to  the  experiment.  Professor  Sidney  Martin  carried  out  a  number  of 
experiments  for  the  Royal  Commission  on  Tuberculosis,!  amongst  which  three  out 
of  four  calves  fed  on  human  tuberculous  sputum  contracted  the  disease. 

Iii  1902  Koch  again  emphasised  the  comparative  rarity  of  primary 
intestinal  tuberculosis  in  the  human  being,  and  the  local,  as 
distinguished  from  the  general,  infective  nature  of  accidental  bovine 
inoculation  of  man  (tuberculosis  verrucosa  cutis).  In  isolated  cases 
the  nearest  lymph  glands  might  become  affected,  but  the  disease 
remained  nevertheless  a  local  one.  Dr  Koch  further  expressed  the 
view  that  if  bovine  tuberculosis  was  transmissible  to  man  by  means 
of  the  milk  of  cows  with  tuberculous  udders,  it  would  be  reasonable 
to  suppose  that  "groups  of  illnesses"  would  occur,  in  a  manner 
analogous  to  other  infective  diseases,  though  the  circumstances  would 
differ  owing  to  the  different  length  of  the  incubation  periods.  By 
way  of  illustrating  the  non-infectivity  of  bovine  tubercle  bacilli 
conveyed  by  milk,  Koch  points  out  (a)  that  bovine  tubercle  bacilli 
must  be  taken  into  the  human  system  very  frequently,  as  1  to  2 
per  cent,  of  all  milch  cows  surfer  from  tuberculous  udders ;  (b)  that 
in  addition  to  being  drunk  in  considerable  quantity  and  for  long 
periods,  such  milk  is  also  widely  distributed;  (c)  that  domestic 
sterilisation  of  milk  does  not  occur  to  any  appreciable  extent ;  (d) 
that  the  same  may  be  said  of  the  large  dairies ;  and  finally  (e)  that 
if  milk  under  such  circumstances  is  dangerous,  the  butter  derived 
from  it  will  also  be  dangerous.  For  these  reasons  he  maintained 
that  any  resulting  disease  must  be  widespread.  Yet  Koch  has  found 
"instead  of  the  countless  cases,"  which  we  ought  to  expect,  "two 
groups  of  illnesses  and  28  isolated  cases  of  illness."  On  examina- 
tion he  finds  most  of  these  recorded  cases  not  free  from  objection. 
To  carry  conviction  as  to  milk-borne  tuberculosis,  Koch  maintains, 
that  the  following  conditions  must  be  fulfilled: — (i.)  Certain  proof 
of  tubercle  in  the  person  affected;  (ii.)  exclusion  of  other  sources 
of  infection ;  (iii.)  the  condition  of  all  the  consumers  of  the  suspected 
milk;  (iv.)  the  exact  source  of  the  suspected  milk,  particularly  in 
respect  to  the  disease  of  the  udder  of  the  cow  yielding  the  milk. 
Finally,  he  concludes  that  all  that  can  be  said  at  present  is  that  the 
injurious  effects  of  rnilk  infected  with  bovine  tuberculosis  and  its 
products  are  not  proven. 

On  the  other  hand,  many  other  workers  have  been  investigating 

*  Bacterioloyi/  and  Infective  Diseases— Edgar  M.  Crookshank,  1896,  pp.  389-391. 

f  Report  of  the  Royal  Commission  appointed  to  inquire  into  the  Effect  of  Food 
derived  from  Tuberculous  Animals  on  Human  Health,  1895,  part  iii.,  Appendix, 
pp.  18  and  19. 


INTEkCOMMUNICABlLlTY  343 

iho  matter  in  Europe  and  America,  and  Delepine,*  Hamilton/)"  Ortli, 
and  Behring  J  are  amongst  those  who  have  obtained  positive  results. 
Hamilton  was  able  again  to  establish  the  truth  of  Martin's  statement, 
that  not  infrequently  tuberculosis  occurred  in  animals  fed  on 
tubercular  sputum  without  affecting  the  inesenteric  and  other 
intestinal  glands  upon  which  Koch  relied  as  indication  of  positive 
results. 

The  fundamental  feature  of  Behring's  theory  based  upon  his 
experiments,  the  results  of  which  are  entirely  opposed  to  those  of 
Koch,  is  that  tuberculosis  in  animals  and  in  human  beings 
represents  different  varieties  of  the  same  disease,  and  that  it  is 
transferable,  especially  by  the  agency  of  tuberculous  milk.  He 
distinguishes  in  this  respect  between  adults  and  infants,  and  main- 
tains* that  while  the  former,  except  under  special  conditions  of  the 
digestive  organs,  may  safely  partake  of  unsterilised  milk,  infants 
are  particularly  liable  to  infection  from  this  source.  Experiments 
made  on  newborn  foals,  calves,  guinea-pigs,  and  other  animals  show 
that  the  mucous  membrane  of  the  intestines  at  that  stage  of  their 
development  is  like  "  a  filter  with  very  large  pores,"  and  that  the 
bacilli  of  infection  pass  through  it  into  the  blood  precisely  as  if  the 
animals  had  been  inoculated  with  the  poison.  In  subsequent  stages 
of  their  development  these  animals  are  provided  by  nature  with  a 
mucous  membrane  which  tends  to  exclude  the  danger  of  infection. 
Behring  is  convinced  that  the  same  holds  true  of  infants  and  adults, 
and  that  a  large  portion  of  mankind  is  infected  in  infancy  with  the 
germs  of  tuberculosis  derived  from  cows'  milk.  In  support  of  his 
assertion  he  adduces  statistics  both  of  anatomical  and  of  pathological 
investigation. 

This  latter  evidence  tending  to  show  the  transmission  of  tuber- 
culosis to  man  by  means  of  milk  and  meat,  is  of  the  same  character 
as  that  upon  which  the  Eoyal  Commission  relied  when  it 
reported : — "  We  cannot  refuse  to  apply,  and  we  do  not  hesitate  to 
apply,  to  the  case  of  the  human  subject,  the  evidence  (of  trans- 
mission of  the  disease)  thus  obtained  from  a  variety  of  animals 
that  differ  widely  in  their  habits  of  feeding — herbivora,  carnivora, 
omnivora.  As  regards  man,  we  must  believe  that  any  person 
who  takes  tuberculous  matter  into  the  body  as  food  incurs  some 
risk  of  acquiring  tuberculous  disease."  §  And  again,  "  We  have 
obtained  ample  evidence  that  food  derived  from  tuberculous  animals 
can  produce  tuberculosis  in  healthy  animals.  In  the  absence  of 

*  Brit.  Med.  Jour.,  1901,  ii.,  p.  1224. 

t  Trans,   of  Highland  and  Agricult.  Sac.  of  Scotland,  1903,  and  Public  Health, 
1903,  p.  689. 

j  Deut.  Med.  Woch.,  1903. 

§  Report  of  Eoyal  Commission,  1895,  part  i.,  p.  10,  par.  22. 


344    TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

direct  experiments  on  human  subjects  we  infer  that  man  also  can 
acquire  tuberculosis  by  feeding  upon  materials  derived  from  tuber- 
culous food-animals."  * 

Viewing  all  the  facts,  there  can  be  little  doubt  but  that  this  con- 
clusion is  the  right  one  from  the  point  of  view  of  the  public  health. 
Various  circumstances  have  in  all  probability  contributed  to  render 
unsuccessful  or  irregular  in  result  the  numerous  feeding  experiments 
which  have  been  made.  The  tissues  of  animals  differ  greatly  in 
susceptibility  to  tuberculosis;  the  infective  material  is  exposed  to 
the  digestive  juices  which  are,  in  measure,  germicidal,  and  yet  not 
equally  so ;  the  virulence  of  the  infective  material  itself  varies 
enormously,  as  does  the  virulence  between  different  generations  or 
races  of  tubercle  bacilli.  Hence  it  comes  about  that  one  animal 
may  eat  with  its  food  a  certain  amount  of  tuberculous  material,  and 
yet  not  develop  tuberculosis,  whilst  another  animal  of  the  same 
species  might  quickly  develop  the  disease,  which  would  in  all 
probability  show  itself  at  the  animal's  weakest  point,  and  not 
always  necessarily  in  the  intestine.  Further,  there  is  another  point 
which  should  not  be  overlooked,  namely,  the  subsequent  treatment 
of  the  inoculated  animal.  Whilst  it  is  essential  to  prove  that  the 
animal  to  be  inoculated  is  free  from  tuberculosis,  it  should  be 
remembered  that  in  taking  very  healthy  animals  for  experiment, 
and  in  subsequently  treating  them  in  what  may  be  termed  an 
"ideal"  fashion,  some  of  the  very  conditions  essential  to  the  pro- 
duction of  the  disease  in  ordinary  life  are  removed.  As  in  men,  so 
in  cattle  and  other  animals,  it  may  be  presumed  that  abundance  of 
good  food  and  fresh  air,  and,  in  general,  an  ideal  environment,  tend 
to  counteract  the  effect  of  the  inoculated  or  communicated  virus. 
Thus  such  experiments  as  those  stated  above  may  not  always  fairly 
represent  the  modes  of  transmission  of  the  disease  as  they  occur 
in  ordinary  life.  It  is  not  the  "very  healthy"  animal  of  a  herd, 
well  housed  and  fed,  which  contracts  tuberculosis. 

As  a  result  of  the  wide  differences  of  opinion  revealed  by  the 
pronouncement  of  Koch's  views,  special  Commissions  of  Inquiry  were 
instituted  in  Germany,  Great  Britain,  and  other  countries,  in  addition 
to  the  individual  research  work  to  which  reference  has  been  made. 
As  this  book  has  been  passing  through  the  press,  reports  of  these 
inquiries  have  been  made  by  the  German  Imperial  Health  Office  and 
the  Royal  Commission  on  Tuberculosis,  appointed  in  1901  by  the 
British  Government.  The  conclusions  are  briefly  as  follows : — 

Kossel,  Weber,  and  Heuss,  who  carried  out  a  comparative  research 

upon  tubercle  bacilli  of  different  origins,  made  a  number  of  experiments 

on  calves  by  injecting  some  forty  different  strains  of  human  bacilli 

and  fifteen  strains  from  bovine,  fowl,  and  swine  sources.     They  con- 

*  Report  of  Royal  Commission,  1895,  part  i.,  p.  20,  par.  77. 


1NTERCOMMUNICABIL1TY  345 

elude  as  a  result  of  these  experiments  that  in  a  preponderating 
number  of  cases  of  human  tuberculosis,  tubercle  bacilli  were  found 
distinguishable  from  the  bovine  bacilli  of  Perlsucht  in  cows 
morphologically,  culturally,  and  in  pathogenic  properties,  but  that 
exceptionally  in  man  tubercle  bacilli  occur  which  cannot  be  distin- 
guished. They  hold,  nevertheless,  that  the  possibility  of  infection  in 
man,  under  certain  circumstances,  by  milk  from  tuberculous  udders  is 
proved.  They  found  that  generalised  tuberculosis  was  produced  in 
animals  by  injecting  strains  of  tubercle  bacilli  obtained  from  tuber- 
culous diseases  in  children.* 

The  Eoyal  Commission  appointed  in  this  country  has  also  issued 
an  interim  report  (1904),  signed  by  Sir  M.  Foster  and  Professors  Sims 
Woodhead,  Sidney  Martin,  MacFadyean,  and  Boyce.  The  Commission 
was  appointed  to  inquire  and  report  with  respect  to  tuberculosis : — 

(1)  Whether  the  disease  in  animals  and  man  is  one  and  the  same ; 

(2)  whether  animals  and  man  can  be  reciprocally  infected  with  it; 
and   (3)  under  what  conditions,  if  at  all,   the   transmission   of   the 
disease  from  animals  to  man  takes  place,  and  what  are  the  circum- 
stances favourable  or  unfavourable  to  such  transmission. 

The  first  line  of  inquiry  upon  which  they  entered  may  be  stated 
in  their  own  words,  as  follows : — 

What  are  the  effects  produced  by  introducing  into  the  body  of  the  bovine  animal 
(calf,  heifer,  cow),  either  through  the  alimentary  canal  as  food,  or  directly  into  the 
tissues  by  subcutaneous  or  other  injection,  tuberculous  material  of  human  origin, 
i.e.,  material  containing  living  tubercle  bacilli  obtained  from  various  cases  of  tuber- 
culous disease  in  human  beings,  and  how  far  do  these  effects  resemble  or  differ  from 
the  effects  produced  by  introducing  into  the  bovine  animal,  under  conditions  as  similar 
as  possible,  tuberculous  material  of  bovine  origin,  i.e.,  material  containing  living 
tubercle  bacilli  obtained  from  cases  of  tuberculous  disease  in  the  cow,  calf,  or  ox  ? 

We  have  up  to  the  present  made  use,  in  the  above  inquiry,  of  more  than  twenty 
different  **  strains  "  of  tuberculous  material  of  human  origin,  that  is  to  say,  of  material 
taken  from  more  than  twenty  cases  of  tuberculous  disease  in  human  beings,  including 
sputum  from  phthisical  patients,  and  the  diseased  parts  of  the  lungs  in  pulmonary 
tuberculosis,  mesenteric  glands  in  primary  abdominal  tuberculosis,  tuberculous 
bronchial  and  cervical  glands,  and  tuberculous  joints.  We  have  compared  the 
effects  produced  by  these  with  the  effects  produced  by  several  different  strains  of 
tuberculous  material  of  bovine  origin. 

In  the  case  of  seven  of  the  above  strains  of  human  origin,  the  introduction  of  the 
human  tuberculous  material  into  cattle  gave  rise  at  once  to  acute  tuberculosis,  with 
the  development  of  widespread  disease  in  various  organs  of  the  body,  such  as  the 
lungs,  spleen,  liver,  lymphatic  glands,  etc.  In  some  instances  the  disease  was  of 
remarkable  severity. 

In  the  case  of  the  remaining  strains,  the  bovine  animal  into  which  the  tuberculous 
material  was  first  introduced  was  affected  to  a  less  extent.  The  tuberculous  disease 
was  either  limited  to  the  spot  where  the  material  was  introduced  (this  occurred,  how- 
ever, in  two  instances  only,  and  these  at  the  very  beginning  of  our  inquiry),  or  spread 
to  a  variable  extent  from  the  seat  of  inoculation  along  the  lymphatic  glands,  with,  at 
most,  the  appearance  of  a  very  small  amount  of  tubercle  in  such  organs  as  the  lungs 
and  spleen.  Yet  tuberculous  material  taken  from  the  bovine  animal  thus  affected, 
and  introduced  successively  into  other  bovine  animals,  or  into  guinea-pigs  from  which 

*  Tuberkulose-Arbeiten  aus  dem  Kaiserlichen  Gesundheitsamte,  Heft  i.,  1904,  p.  34. 


346   TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

bovine  animals  were  subsequently  inoculated,  has,  up  to  the  present,  in  the  case  of 
five  of  these  remaining  strains,  ultimately  given  rise  in  the  bovine  animal  to  general 
tuberculosis  of  an  intense  character ;  and  we  are  still  carrying  out  observations  in 
this  direction. 

We  have  very  carefully  compared  the  disease  thus  set  up  in  the  bovine  animal  by 
material  of  human  origin  with  that  set  up  in  the  bovine  animal  by  material  of  bovine 
origin,  and  so  far  we  have  found  the  one,  both  in  its  broad  general  features  and  in 
its  finer  histological  details,  to  be  identical  with  the  other.  We  have  so  far  failed  to 
discover  any  character  by  which  we  could  distinguish  the  one  from  the  other ;  and 
our  records  contain  accounts  of  the  post-mortem  examinations  of  bovine  animals 
infected  with  tuberculous  material  of  human  origin,  which  might  be  used  as  typical 
descriptions  of  ordinary  bovine  tuberculosis. 

The  results  which  we  have  thus  obtained  are  so  striking,  that  we  have  felt  it  our 
duty  to  make  them  known,  without  further  delay,  in  the  present  interim  report 

The  Commission  defer  to  a  further  report  all  narration  of  the 
details  of  their  experiments,  as  well  as  all  discussions,  including  those 
dealing  with  the  influence  of  dose  and  of  individual  as  well  as  racial 
susceptibility,  with  questions  of  the  specific  virulence  of  the  different 
strains  of  bacilli,  with  the  relative  activity  of  cultures  of  bacilli  and 
of  emulsions  of  tuberculous  organs  and  tissues,  and  with  other  points. 

Meanwhile  they  have  thought  it  their  duty  to  make  this  short 
interim  report,  for  the  reason  that  the  result  at  which  they  have 
arrived,  namely,  that  tubercle  of  human  origin  can  give  rise  in  the 
bovine  animal  to  tuberculosis  identical  with  ordinary  bovine  tubercu- 
losis, seems  to  them  to  show  quite  clearly  that  it  would  be  most  unwise 
to  frame  or  modify  legislative  measures  in  accordance  with  the  view 
that  human  and  bovine  tubercle  bacilli  are  specifically  different  from 
each  other,  and  that  the  disease  caused  by  the  one  is  a  wholly  different 
thing  from  the  disease  caused  by  the  other. 

In  this  final  conclusion  as  to  administrative  measures  both  German 
and  British  Commissions  agree.  They  also  agree  as  to  the  inter- 
communicability  of  bovine  and  human  tuberculosis.* 

Diagnosis  of  Bovine  Tuberculosis.— There  are  three  methods 
of  diagnosis — clinical,  bacteriological,  and  by  means  of  tuberculin. 

(a)  Clinical. — When  tuberculosis  affects  the  lungs  and  respiratory 
organs  generally,  it  is  accompanied  by  a  frequent  cough,  but  no  fever. 
There  is  disturbance  of  the  respiration,  the  breathing  being  quickened 
by  slight  exertion  or  excitement,  and  the  cough  stimulated  by 
changes  of  temperature.  The  departure  from  the  normal  in  the 
relative  length  of  the  inspiratory  and  expiratory  movement  (the 
expiration  being  markedly  prolonged)  can  be  readily  seen  as  a  rule, 
and  not  uncommonly  in  these  cases  a  rough  harsh  sound  may  be 
heard  in  the  throat  during  respiration.  By  auscultation  it  is  possible 
sometimes  to  detect  dull  portions  of  the  lung  surrounded  by  areas  of 
increased  resonance.  The  vesicular  murmur  is  louder  and  harsher 

*  This  subject  is  fully  discussed  in  Bull.  53  (1904),  of  U.S.  Dept.  of  Agriculture 
(Salmon). 


DIAGNOSIS  OF  BOVINE  TUBERCULOSIS  347 

than  normal  over  the  tipper  half  of  the  chest,  and  is  particularly 
marked  during  expiration.  Usually  the  superficial  glands  in  the  throat, 
those  between  the  jaws,  and  under  the  ear  or  of  the  udder  are  swollen 
and  hard.  The  animal  may  continue  for  months  in  an  apparently 
healthy  condition.  When  the  disease  is  abdominal  and  the  glands 
and  organs  in  the  belly  are  chiefly  affected,  the  symptoms  of  defective 
nutrition  are  early  evident,  namely,  emaciation,  lessened  milk  secre- 
tion, indigestion,  breathlessness,  and  more  or  less  rapid  failure  in 
general  health.  In  these  cases  the  udder  should  be  especially 
examined. 

It  should  be  noted  that  clinical  diagnosis  of  tuberculosis,  especi- 
ally of  udder  tuberculosis,  does  not  enable  us  to  judge  whether 
tubercle  bacilli  are  secreted  along  with  the  milk  of  the  cow  in 
question.  The  bacteriological  test  and  tuberculin  are  necessary. 
The  former  is  sometimes  tedious,  and  the  latter  remains  at  present 
our  only  quick  and  sure  method. 

(5)  Bacteriological  Examination. — This  method  of  diagnosis  can 
be  applied  at  once  in  suspected  udder  disease  by  examination  of  the 
milk ;  or,  as  recommended  by  Nocard,  a  trocher  may  be  used  by 
which  a  small  fragment  of  tissue  from  the  indurated  portion  of  the 
udder  may  be  obtained  for"  examination.  Mucus  or  discharges  from 
throat,  wounds,  and  ulcers  may  also  be  examined  and  assist  in 
diagnosis.  The  only  sure  method  of  bacteriological  examination  is 
by  inoculation  of  animals.  Microscopical  and  cultural  tests  are 
unreliable. 

(c)  Tuberculin. — In  recent  years  the  method  of  testing  herds  for 
tuberculosis  by  means  of  tuberculin  has  come  into  vogue,  and  it  will 
be  necessary  to  refer  briefly  to  this  subject.  The  discovery  by  Koch, 
in  1890,  of  the  production  of  fever,  indicated  by  a  rise  in  tempera- 
ture, in  tuberculous  animals  into  which  he  injected  a  sterilised 
glycerine  extract  of  pure  cultures  of  tubercle  bacilli,  while  it 
produced  no  effect  whatever  when  the  animals  were  free  from  that 
disease,  furnished  us  with  a  simple  but  fairly  reliable  diagnostic 
agent. 

Tuberculin  is  a  soluble  product  of  cultures  of  tubercle  bacilli,  of 
which  a  glycerine  extract  is  made,  which  is  sterilised  by  heat  and 
filtered  through  porcelain,  so  that  it  contains  no  living  germs,  and 
therefore  cannot  produce  tuberculosis  in  animals  injected  with  it. 
It  has,  therefore,  no  effect  on  healthy  animals  ;  in  some  cases  the 
disease  is  aggravated  by  it  when  it  exists,  but  it  cannot  be  produced 
by  it.  The  lymph  must  not  be  exposed  to  sunlight ;  it  must  not  be 
frozen,  and  must  be  kept  well  corked  to  exclude  air. 

Koch's  "old  tuberculin"  is  made  from  glycerine-veal  broth 
cultures  of  B.  tuberculosis  by  means  of  evaporation  and  precipitation 
with. alcohol.  The  liquid  cultures  are  thus  concentrated  to  one-tenth 


348    TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

of  their  original  bulk,  and  then  passed  through  a  Chamberland  filter. 
The  brown  and  viscid  filtrate  is  the  tuberculin.  Buchner  and 
Eomer  pointed  out  that  the  proteins  of  other  bacteria  have  a  similar 
effect  upon  tuberculosis,  that  is,  cause  a  reaction  with  rise  of 
temperature.  In  1897,  Koch  was  able  to  improve  his  tuberculin, 
and  under  the  name  "Tuberculin  T.  B."  recommended  a  new 
preparation.  In  point  of  fact,  the  new  preparation  takes  three 
forms,  distinguished  by  the  letters  T.  A.  (alkaline  tuberculin),  T.  0. 
(upper  tuberculin,  Germ,  ober),  and  T.  E.  (residual  tuberculin).  T.  A. 
is  extracted  from  a  young  and  virulent  culture  of  B.  tuberculosis  by 
means  of  a  one-tenth  normal  solution  of  caustic  soda,  and  the 
solution  is  filtered.  The  reaction  on  inoculation  is  intense,  and  may 
be  accompanied  with  abscesses.  Accordingly,  its  clinical  use  is 
open  to  objection.  T.  0.  and  T.  E.  are  prepared  by  vigorously 
pounding  in  a  mortar  dried  cultures  of  the  tubercle  bacilli  and  then 
adding  distilled  water.  The  emulsion  is  thoroughly  centrifugalised. 
The  clear,  opalescent  fluid  collecting  at  the  upper  part  of  the  tube 
contains  no  tubercle  bacilli,  and  constitutes  in  the  first  centrifugal- 
isation  T.  0.  The  debris  or  residuum  of  tubercle  bacilli  remaining  at 
the  bottom  of  the  tube  is  used  for  the  production  of  T.  E.  This 
residue  is  dried,  triturated  with  distilled  water,  and  centrifugalised 
repeatedly  until  hardly  any  residue  remains.  Twenty  per  cent,  of 
glycerine  is  then  added  to  both  preparations  for  purposes  of  pre- 
servation. T.  E.  alone  is  used  clinically. 

The  method  of  use  is  as  follows :  The  animals  are  kept  a  day  or 
two  in  their  byres,  and  the  temperature  is  taken  to  standardise  the 
normal,  which  is  generally  about  102'2°  F.  The  tuberculin  is  then 
injected  (30-40  centigrammes),  and  if  the  animal  be  tuberculous, 
there  is  a  rise  in  temperature  of  1J°  to  3°.  The  fever  usually  begins 
between  the  twelfth  and  fifteenth  hour  after  injection,  and  lasts 
several  hours.  The  more  nearly  the  temperature  approaches  104°  F., 
the  more  reason  is  there  to  suspect  tuberculosis  (Bang).  The  dura- 
tion and  intensity  of  the  reaction,  however,  has  not  a  direct  relation 
to  the  number  or  gravity  of  the  lesions,  but  the  same  dose  in  healthy 
cattle  causes  no  appreciable  febrile  reaction.  The  tuberculous  calf 
reacts  just  as  well  as  the  adult,  but  the  dose  used  is  generally 
smaller. 

Tuberculin  injection  has  no  bad  effects  on  the  secretion  of  milk, 
either  in  quantity  or  in  quality.  The  consensus  of  opinion  of  those 
most  experienced  is  that  it  does  not  lessen  the  secretion  of  milk  in 
dairy  cattle,  consequently  they  may  be  tested  even  when  in  full 
milk  without  disturbing  its  secretion,  unless  it  be  during  the  few 
hours  of  its  absorption.  It  does  not  cause  abortion  in  cows,  or 
sterility  in  bulls. 

It  is  the  quickest  and   most   reliable   method  of   diagnosis  of 


IN  PIG  AND  SHEEP  349 

bovine  tuberculosis  which  we  possess.  Schlitz  maintains  that 
failures  in  diagnosis  by  tuberculin  injection  only  amount  to  2*9  per 
cent.  Obviously,  it  may  fail  in  animals  which  are  highly  tuberculous, 
owing  to  the  fact  that  their  tissue  already  contains  so  much  tuberculin 
that  they  are  unable  to  respond  any  longer  to  the  tuberculin  test. 
If  there  is  any  lesion  whatever  in  the  udder,  and  there  is  a  reaction 
to  tuberculin,  the  milk  from  that  cow  should  not  be  used.* 

Tuberculosis  of  Other  Animals 

Tuberculosis  of  the  Pig*  is  less  common  than  that  of  cattle,  but 
not  so  rare  as  that  of  the  calf  (Nocard).  In  nine  out  of  ten  cases 
the  pig  is  infected  by  ingestion,  particularly  when  fed  on  the  refuse 
from  dairies  and  cheese  factories.  The  disease  follows  the  same 
course  as  in  cattle,  but  generalisation  is  more  common  and  more 
rapid.  Lesions  of  the  abdominal  organs  occur  in  almost  every  case. 
The  glands,  particularly  those  of  the  throat,  are  markedly  affected. 
The  finding  of  the  tubercle  bacillus  is  difficult,  and  the  only  safe 
test  is  inoculation.  The  massive  lesions  are  often  thinly  scattered, 
rich  in  giant  cells,  and  containing  few  bacilli.  The  disease  usually 
assumes  the  acute  or  "  galloping  "  form,  and  not  infrequently  emacia- 
tion is  absent  and  the  dead  pork  meat  possesses  a  healthy  and  fat 
appearance.  The  internal  organs  and  glands  are  the  chief  sites  of 
disease.  The  whole  carcase  should  be  condemned.  In  a  pork  carcase 
seized  by  the  writer  in  Finsbury  in  1904,  the  retro-pharyngeal, 
submaxillary,  cervical,  and  mediastinal  glands  were  enormously 
enlarged  and  caseous,  the  deep  glands  of  the  body  and  the  udder 
and  its  glands  were  also  affected,  and  the  joints  of  the  right  fore 
foot  were  ankylosed  owing  to  tuberculous  infiltration.  The  internal 
organs  in  this  case  were  a  mass  of  tubercle. 

Sheep  are  very  rarely  affected  with  tuberculosis,  though  there 
is  evidence  which  goes  to  prove  that  very  long  confinement  in  limited 
space  with  tuberculous  cattle  might  result  in  transmitting  tuber- 
culosis to  sheep.  One  of  the  few  cases  on  record  in  this  country 
was  met  with  by  the  writer,  and  has  been  described  by  Foulerton.'|- 
This  was  a  half-bred,  emaciated  ewe  having  both  lungs  extensively 
consolidated,  and  containing  numerous  tubercles  which  were  also 
present  on  the  pleurae.  The  liver,  spleen,  both  kidneys,  and  lymphatic 
glands  were  affected.  An  emulsion,  made  from  one  of  the  affected 
glands,  inoculated  into  the  guinea-pig,  caused  death  from  generalised 
tuberculosis.  The  sheep,  though  rarely  attacked,  is  not  naturally 
immune. 

*  For  a  discussion  as  to  the  practical  use  of  the  tuberculin  test,  see  Trans.  Brit. 
Cong,  on  Tuberculosis,  1901,  vol.  ii.,  pp.  235-78  (Delepine). 

T  Transactions  of  the  Pathological  Society  of  London,  1902,  vol.  liii.,  pt.  iii.,  p.  428. 


350    TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

Tuberculosis  in  the  Horse  is  relatively  very  rare.  It  attacks 
the  organs  of  the  abdominal  cavity,  especially  the  glands ;  it  affects 
the  lung  secondarily  as  a  rule.  The  cases  are  generally  isolated  ones, 
even  though  the  animal  belongs  to  a  stud.  Nocard  holds  that  the 
bacillus  obtained  from  the  pulmonary  variety  is  like  the  human 
type,  whilst  the  abdominal  variety  is  more  like  the  avian  bacillus. 

Dog*. — Nbcard  says:  "If  the  dog  can  become  tuberculous  from 
contact  with  man,  the  converse  is  equally  true.  Infection  is  at  any 
rate  possible  when  a  house-dog  scatters  on  to  the  floor,  carpet,  or 
bed,  during  its  fit  of  coughing,  virulent  material,  which  is  rendered 
extremely  dangerous  by  drying,  especially  for  children,  its  habitual 
playmates.  The  most  elementary  prudence  would  recommend  the 
banishment  from  a  room  of  every  dog  which  coughs  frequently,  even 
though  it  only  seems  to  be  suffering  from  some  common  affection  of 
the  bronchi  or  lung."  * 

Birds. — Tuberculosis  is  a  common  disease  among  birds  of  the 
poultry-yard:  poultry,  pigeons,  turkeys,  pea-fowl,  guinea-fowl,  etc. 
They  are  infected  almost  exclusively  through  the  digestive  tract, 
generally  by  devouring  infected  secretions  or  organs  of  previous 
tubercular  fowls,  and  though  very  susceptible  in  this  way,  birds  can 
consume  large  quantities  of  phthisical  sputum  without  becoming 
tubercular.  Whatever  the  position  or  form  of  avian  tuberculosis,  the 
bacilli  are  present  in  enormous  numbers,  and  are  often  much  shorter 
but  sometimes  longer  than  those  met  with  in  tuberculous  mammalia, 
and  grow  outside  the  body  at  a  higher  temperature  (43°  C.).  They  are 
said  also  to  be  more  resistant  and  of  quicker  growth.  The  species 
is  probably  identical  with  Koch's  bacillus,  though  there  are  differences 
(Maffucci).  In  the  nodule,  which  is  larger  than  in  human  tuber- 
culosis, there  are  few  or  no  giant  cells,  and  it  does  not  so  readily 
break  down.  Guinea-pigs  and  other  animals  are  not  so  readily 
infected  with  avian  tubercle  as  mammalian.  The  writer  has  prepared 
a  number  of  histological  specimens  illustrating  the  comparative 
pathology  of  tuberculosis,  particularly  in  birds  which  have  died  of 
the  disease  in  the  Zoological  Gardens,  London,  including  guan,  quail, 
ostrich,  rhea,  currasow,  swan,  cuckoo,  vulture,  goose,  eagle,  fowl, 
pheasant,  parrot,  etc.  In  most  cases  the  disease  affects  the  organs 
of  the  alimentary  canal,  especially  the  liver.  The  lungs  are  rarely 
affected  except  secondarily.  The  disease  frequently  develops  rapidly 
like  an  acute  infective  disease,  and  the  bacilli  may  often  be  found  in  the 
tissues  arranged  in  large  colonies,  as  in  leprosy  in  the  human  tissues. 

The   bacillus   of  avian   tubercle    differs   from   the   organism    of 

tuberculosis  in  mammals   in   the  appearance   of   the   cultures  and 

the  temperature  conditions.      Fischel,  who  worked  under  Huppe's 

direction,  says  both  micro-organisms  are  of  one  and  the  same  kind 

*  Animal  Tuberculosis,  p.  129. 


PLATE  24. 


S     2 


IN  BIRDS  AND  COLD-BLOODED  ANIMALS  351 

as  regards  nutritive  media.  In  consequence  of  the  different  physio- 
logical nutritive  media  of  the  colder  mammalian  bodies  on  the  one 
hand,  and  the  warmer  avian  bodies  on  the  other,  a  distinction  has 
arisen  between  the  two  kinds.  By  artificial  cultivation  Fischel 
succeeded  in  bringing  about  approximation  in  outer  qualities  between 
the  two  bacilli ;  he  succeeded  in  getting  the  organism  of  tuberculosis 
in*  mammals  accustomed  to  a  higher  temperature,  and  in  given 
nutritive  media  he  obtained  a  resemblance  in  the  appearance  of  the 
cultures,  but  as  regards  pathogenesis  he  could  not  transfer  one  to 
the  other.  Fischel  states  that  he  was  able,  with  the  organism  of 
avian  tuberculosis,  to  bring  about  a  general  tuberculosis  in  a  guinea- 
pig,  but  the  cultures  started  out  of  the  organs  of  this  animal  were 
not  identical  with  those  of  avian  tuberculosis. 

Tuberculosis  in  cold-blooded  animals  is  in  the  same  way  to  be 
regarded  as  a  modification  of  the  tuberculosis  in  mammals.  Bataillon 
and  Terre  succeeded  in  cultivating  tuberculosis  in  mammals  and 
birds  by  means  of  passing  it  through  the  body  of  a  frog  at  room 
temperature.  Lubarsh  was  able  to  modify  tuberculosis  in  mammals 
by  passage  through  the  body  of  a  frog,  so  that  cultures  taken  from 
the  spleen  of  a  frog  grew  at  a  temperature  of  as  much  as  28-30°. 
Dubard  also  cultivated  cultures  taken  from  fish,  inoculated  with 
mammalian  tuberculosis  which  also  thrived  at  room  temperature. 
Moeller  was  able  to  produce  cultures  from  the  spleen  of  a  slow-worm 
inoculated  with  sputum  containing  tubercle  bacilli,  which  flourished 
at  20°;  but  which  ceased  to  grow  at  a  temperature  of  30°  and  over. 
The  cultures  resemble  in  appearance  those  of  avian  tuberculosis,  grow 
at  room  temperature,  and  are  moist  and  thick. 

It  should  be  added  that  certain  abnormal  and  tubercle-like 
conditions  have  been  met  with  in  the  carp,  and  from  such  conditions 
a  bacillus  morphologically  and  tinctorially  similar  to  the  tubercle 
bacillus  has  been  isolated. 

Most  bacteriologists  maintain  that  the  B.  tuberculosis  of  Koch  is 
the  common  denominator  in  all  tubercular  disease,  whatever  and 
wherever  its  manifestations,  in  all  animals.  The  bacillus,  they 
hold,  may,  however,  experience  profound  modifications  owing  to 
successive  passages  through  the  bodies  of  divers  species  of  animals. 
But  if  the  modifications  which  it  undergoes  as  a  result  of  trans- 
mission through  birds,  for  example,  are  profound  enough  to  make 
the  bacillus  of  avian  tubercle  a  peculiar  variety,  though  not  a  distinct 
and  separate  species,  of  Koch's  bacillus,  they  are  not  enough,  it  is 
generally  believed,  to  make  these  bacilli  two  Distinct  species.  We 
may,  therefore,  take  it  for  granted  that  tuberculosis  is  one  and  the 
same  disease,  generically,  with  various  manifestations,  common  to 
man  and  animals,  intercommunicable,  and  having  but  one  vera  causa, 
the  B.  tuberculosis  of  Koch. 


352     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

The  Prevention  of  Tuberculosis 

At  the  present  time  much  attention  is  being  directed  to  the 
administrative  and  personal  control  of  tuberculosis.  How  greatly 
this  is  needed  in  so  preventable  a  disease  is  evident  from  a  perusal 
of  the  following  table  from  the  Kegistrar-G-eneral's  reports : — 


ENGLAND  AND  WALES 
ANNUAL  DEATH-RATES  FROM  ALL  TUBERCULAR  DISEASES 

The  following  is  a  table  of  death-rates  to  a  million  living  (England 
and  Wales),  1880-1901  (Reg.-Gen.  Annual  Reports):— 


1880 

1881 

1882 

1883 

1884 

1885 

1886 

1887 

1888 

1889 

1890 

Tabes  Mesenterica     . 
Tubercular  Meningitis 
Phthisis      .... 
Other  Forms 

370 
330 
1869 
129 

284 
276 
1825 
145 

313 

264 
1850 
153 

289 
262 

1880 
160 

310 
264 
1827 
170 

251 
253 

1770 
157 

300 
257 
1739 
177 

253 
236 
1615 

179 

240 
239 
1568 
174 

2221 

269 
234 
1573 
183 

265 
240 
1682 
189 

Total     .... 

2698 

2530 

2580 

2591 

2571 

2431 

2473 

2283 

2259 

2376 

1891 

1892 

1893 

1894 

1895 

1896 

1897 

1898 

1899 

1900 

1901 

Tabes  Mesenterica     . 
Tubercular  Meningitis 
Phthisis      .... 
Other  Forms 

251 
247 
1599 
203 

242 
227 
1468 
199 

265 
226 
1468 
186 

192 
211 
1385 
185 

243 

222 
1398 
200 

196 
210 
1307 
179 

201 
213 
1341 
175 

202 
213 
1317 

184 

198 
203 
1336 
174 

185 
198 
1333 

186 

188 
183 
1264 
172 

Total     .... 

2300 

2136 

2145 

1973 

2063 

1892 

1930 

1916 

1911 

1902 

1807 

Tabes  mesenterica  is  a  term  applied  to  tuberculosis  of  the  alimentary  canal  and 
mesenteric  lymph  glands. 

Tubercular  meningitis  is  the  name  of  the  same  disease  as  it  affects  the  mem- 
branes of  the  brain  (acute  hydrocephalus). 

Phthisis  is  the  term  applied  to  "  consumption,"  or  tubercle  in  the  lungs. 


These  figures  show  a  marked  decline  in  the  three  worst  forms  of 
the  disease.     But  this  decline  is  less  marked  in  tabes  mesenterica 


PREVENTION  OF  TUBERCULOSIS  353 

than  in  phthisis  or  tubercular  meningitis,  i.e.  less  in  the  kind  of 
tubercle  located  in  the  abdomen  chiefly.  Fortunately,  the  State  is 
beginning  to  realise  its  duty  in  regard  to  preventive  measures.  The 
abolition  of  private  slaughter-houses,  the  protection  of  meat  and 
milk  supplies,  the  condemnation  of  tuberculous  milch  cows,  and  such- 
like measures,  fall  obviously  within  the  jurisdiction  of  the  State 
rather  than  the  individual,  and  claim  the  earnest  and  urgent  attention 
of  the  public  health  departments  of  Government.* 

Methods  of  Prevention. — A  consideration  of  the  various  facts  set 
forth  in  this  chapter  will  suggest  the  best  means  of  the  prevention  of 
consumption.  These  means  depend  upon  broad  principles  of  sanitation 
and  personal  health  rather  than  on  bacteriological  niceties  or  theore- 
tical considerations.  What  is  required  has  been  stated  briefly  by 
Sir  Hermann  Weber  as: — (a)  Purity  and  free  circulation  of  air;  (6) 
sufficiency  of  good  and  pure  food ;  (o)  well-constructed  and  ventilated 
sunny  rooms  in  houses  situated  on  dry  and  pure  soil;  and  (d)  the 
maintenance  of  the  resisting  power  of  the  body  and  its  different 
organs.f  These  general  desiderata  are  to  be  secured  by  practical 
preventive  methods,  of  which  the  following  are  some  of  the  more 
important : — 

1.  Personal   hygiene   and   the   maintenance  of  a  high  degree  of 
resistance  in  the  human  tissues  is  a  matter  which  must  rest  with  the 
individual  rather  than  the  State,  which  can  only  exert  its  influence, 
generally  speaking,  on  the  environment  of  the  individual.     Below  will 
be  found  a  number  of  particulars  as  to  the  disease,  stated  in  simple 
form,  and  many  of  which  bear  a  direct  relation  to  the  management 
of  personal  conditions.     The  healthy  life  with  sufficient  food,  exercise, 
etc.,  is  what  is  necessary  to  maintain  healthy  tissues.     Mention  may, 
however,  be  made  of  the  abuse  of  alcohol  and  the  neglect  of  simple 
illnesses  as  two  most  powerful  factors  in  the  creation  of  conditions  in 
the  body  favourable  to  tubercle  infection. 

2.  Only   second    in    importance   is    general    sanitation    and    the 
creation  of  a  healthy  environment  for  the  individual  and  the  com- 
munity, and  in  this,  and  subsequent  methods,  much  of  the  preventive 
work  in  tuberculosis  is  centred.     Such  conditions  as  dust  in  the  air, 
overcrowding  in  houses,  too  great  a  density  of  houses  on  the  area,  ill- 
ventilated  and  unclean  rooms  and  workshops,  etc.,  exert  an  indirect 
influence  of  great  force  in  the  propagation  of  tuberculosis,  and  there- 
fore  the  reduction  and   abatement  of   these  conditions  serves  as  a 
means  of  prevention.     The  right  enforcement  of  the  Housing  of  the 
Working  Classes  Acts,  the  Public  Health  Acts,  the  Building  Acts, 

*  See  the  Harben  Lectures,  November,  1898,  by  Sir  Richard  Thome  Thorne, 
Medical  Officer  to  the  Local  Government  Board ;  also  the  Reports  of  the  Royal  Com- 
missions on  Tuberculosis,  1896,  1898,  and  1903. 

I  Tuberculosis,  1899,  vol.  i.,  p.  9. 

Z 


$54   TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

and  the  Factory  and  Workshop  Act  are  therefore  matters  of  great 
importance  in  the  prevention  of  this  disease. 

3.  Food  has  also  been  shown  to  be  infected  in  a  greater  or  less 
degree  with  the  virus  of  tuberculosis,  and  though  the  disease  is  not 
spread  so  greatly  through  this  channel  as  in  other  ways,  it  is  never- 
theless necessary  to  protect  the  public  from  tuberculous  food,  especially 
meat  and  milk.      The   Public  Health  Acts  (1875  and    1891)   give 
powers  of  seizure  of  diseased  food,  and  the  Dairy,  Milkshops,  and 
Cowsheds  Order  of  1885,  and  its  amendments,  operate  in  the  direction 
of  the  control  of  the  milk  supply.     These  latter  Orders  should  be 
unified,  and  much  more  vigorously  enforced  than  has  been  the  case  in 
the  past. 

4.  Lastly,  there  are  certain  measures  of  great  importance  which 
concern  the  avoidance  of  infection  from  diseased  persons.     The  con- 
sumptive is  the  chief  agent  in  the  spread  of  consumption.     Therefore, 
anything  which  lessens  the  degree  of  his  contagiousness  is  a  means  of 
prevention.      The   first  requirement  is   evidently  knowledge  of  the 
existence  of  cases  of  phthisis,  and  this  may  be  obtained  in  various 
ways,  e.g.,  through   hospitals  or  private  practice,  through  poor-law 
institutions,  or  by  voluntary  or  compulsory  notification.     Voluntary 
notification  was  first  adopted  in  this  country  by  the  Local  Authorities 
in  Brighton,  Manchester,  and  Finsbury,  and  is  now  in  vogue  in  many 
districts.     The  results  are  not  wholly  satisfactory,  but  are  better  than 
no  information  at  all.     Compulsory  notification   has  recently  been 
instituted  for  an  experimental  period  in  Sheffield.*     The  cases  of 
phthisis  being  known,  the  next  steps  are  supervision,  disinfection  of 
sputum,  house,  and  clothes,  and,  if  practicable,  isolation  and  treatment 
of  suitable  cases.     Sanatoria  act  partly  as  therapeutic  agencies,  partly 
as   prophylactic   agencies.      Much   is   now   being   done   in   civilised 
countries  in  these  directions,  and  many  sanitary  authorities  carry  out 
disinfection  regularly,  and  make  various  efforts  to  prevent  consump- 
tives infecting  their  neighbours  or  fellow-workmen. 

Still,  after  all,  the  prevention  of  phthisis  is  in  no  small  degree  a 
matter  of  personal  hygiene  and  precautions  to  be  exercised  by  the 
people  themselves. 

Hence  we  hail  with  satisfaction  the  recent  endeavours  to  educate 
public  opinion.  In  order  to  simplify  this  matter,  we  have  placed 
in  a  footnote  a  series  of  statements  embodying  some  of  the  chief 
facts  which  every  individual  in  our  crowded  communities  should 
know.f 

*  Sheffield  Corporation  Act,  1903,  sect.  45. 

t  1.  Tuberculosis  is  a  disease  mainly  affecting  the  lungs  (consumption,  decline, 
phthisis)  of  young  adults  and  the  bowels  of  infants  (tabes  inesentencd).  It  may 
affect  any  part  of  the  body,  and  its  manifestations  are  very  various.  It  also  affects 
animals,  particularly  cattle,  by  whom  it  may  be  transmitted  to  man. 

2.    Its  direct  cause  is  a  microscopic  vegetable  cell,  known   as  the  B.   tuber- 


PREVENTION  OF  TUBERCULOSIS  355 

Pseudo-tuberculosis 

In  1899  the  Pathological  Society  of  London  urged  that  this  term 
should  be  discarded.     It  is  used  here  not  as  concerned  with  diseases 

culosis,  discovered  by  Koch  in  1882.  This  fungus  requires  to  be  magnified  some 
hundreds  of  times  before  it  can  even  be  seen.  When  it  gains  entrance  to  the 
weakened  body  it  sets  up  the  disease,  which  is  an  infectious,  or  sub-infectious, 
disease,  though  different  in  degree  to  the  infectiousness  of,  say,  measles. 

3.  Trade  influence  and  occupation,  in  some  cases,  undoubtedly  predisposes  the 
individual   to   tubercle.      Cramped   attitudes,  exposure   to   dampness   or  cold,  ill 
ventilation,  and  exposure  to  inhalation  of  dust  of  various  kinds,  all  act  in  this  way. 
In  support  of  the  evil  effect  of  each  of  these  three  conditions  much  evidence  could 
be  produced. 

4.  Overcrowding  has  a   definite  influence  in  propagating  tubercular  diseases. 
The  agricultural  counties  without  large  towns,  like  Worcestershire,  Herefordshire, 
Buckinghamshire,  and  Rutland,  are  the  counties  having  the  lowest  mortality  from 
tuberculosis ;    whilst  the  crowded   populations  in  Northumberland,  South  Wales, 
Lancashire,  London,  and  the  West  Riding  suffer  most.    Speaking  more  particularly, 
the  overcrowded  areas  of  London,  such  as  Southwark,  Shoreditch,  Finsbury,  Hoi- 
born,  and  Central  London  generally,  show  very  high  tubercular  death-rates. 

5.  Tuberculosis  is  not  increasing. — During  the  last  thirty  years  it  has  shown,  with 
few  exceptions,  a  steady  decline  in  all  parts  of  England.     "  Consumption  "  is  most 
fatal  in   comparatively  young  people  (fifteen  to  forty-five  years),  whilst  "  tabes  " 
and  other  forms  of  tubercle  are  fatal  chiefly  to  young  children.     These  forms  have 
not  declined  so  much  as  the  lung  form.    The  mortality  in  consumption  of  males  has 
since  1866  been  in  excess  of  that  of  females.     The  age  of  maximum  fatality  from 
consumption  is  later  than  in  the  past,  which  is  probably  due  to  improved  hygiene 
and  treatment. 

6.  This  decline  lias  been  due  not  to  any  special  repressive  measures — for  few  have 
been  carried  out — but  to  a  general  and  extensive  social  improvement  in  the  life  of 
the  people,  to  an  increase  of  knowledge  respecting  tuberculosis  and  hygiene,  to  an 
enormous  advance  in  sanitation,  and  to  more  efficient  land  drainage. 

7.  Not  all  persons  are  equally  liable  to  consumption,   some  being  much  more 
susceptible  than  others.     We  have  mentioned  the  predisposing  influence  of  certain 
trades.    There  is  also  heredity,  which  acts,  as  we  have  said,  in  transmitting  a 
tubercular  tendency,  rarely,  if  ever,  the  actual  virus  of  the  disease ;  there  is,  thirdly, 
the  debilitating  effect  of  previous  illness  or  chronic  alcoholism ;  there  is,  fourthly, 
the  habitual  breathing  of  stagnant,  polluted  air ;  and,  fifthly,  there  are  the  condi- 
tions of  the  environment,  such  as  dampness  and  darkness  of  the  dwelling.     Such 
influences  as  these  weaken  the  resisting  power  of  the  tissues,  and  thus  afford  a 
suitable  nidus  for  the  bacillus  conveyed  in  milk,  or  by  the  inspiration  of  infected 
dust  or  mucus. 

8.  Consumption  may  be  arrested  if  taken  in  time.     In  cases  where  the  lungs  are 
half  gone,  and  consist  of  large  cavities,  it  is  obvious  that  curability  is  out  of.  the 
question.     But  if  the  disease  can  be  properly  treated  in  its  earliest  stages,  there  is 
considerable  likelihood  of  recovery,  or  at  least  of  arrest  of  the  disease. 

9.  The  breath  is  not  dangerous,  as  far  as  we  know,  but  there  is  danger  from 
discharges  of  any  kind  from  any  infected  part,  whether  lungs  or  bowels  ;  for  such 
discharges,  when  dry,  may  readily  pollute  the  air,  and  either  the  bacilli  or  spores  be 
inhaled  into  the  lungs.     The  breath  itself  in  coughing,  shouting,  etc.,  may  emit  a 
fine  spray  of  mucus  or  saliva,  in  which  the  bacilli  may  be  suspended,  and  thus 
convey  infection. 

10.  The  chief  channels  of  personal  infection  in  the  spread  of  the  disease  amongst  a 
community  are  two:    (a)  dried  tubercular  sputum  or  "cough  spray"  (or  other 
tubercular  discharges);  (6)  infected  milk  or  meat.    As  for  milk  and  meat,  boiling 
the  former  and  thoroughly  cooking  the  latter  will  remove  all  danger.     In  any  case, 
there  is  evidence  to  show  that  infection  from  milk  or  meat  is   nothing  like  so 
common  as  infection  from  sputum  or  mucous  particles  from  a  consumptive  patient. 


356     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

simulating  tuberculosis  (protozoal  infections,  parasitical  disease,  etc.), 
but  to  designate  a  pathological  condition  set  up  by  a  special  group 

11.  The  expectoration  is  infective. — This  is  one   of  the  commonest  modes  of 
infection,  and  to  it  is  held  to  be  due  the  large  amount  of  respiratory  tuberculosis 
(consumption,  phthisis).     The  expectoration  from  the  lungs  must  contain,  from  the 
nature  of  the  case,  a  very  large  number  of  bacilli.     As  a  matter  of  fact,  a  single 
consumptive  individual  can  cough  up  in  a  day  millions  of  tubercle  bacilli.     When 
expectoration  becomes  dry,  the  least  current  of  air  will  disseminate  the  infective 
dust,  which  can  by  that  means  be  readily  reinspired.     Infective  saliva  on  pave- 
ments and  floors,  as  well  as  on  handkerchiefs,  or  even  in  books,  may  thus  become  a 
source  of  danger  to  others.     The  discharges  from  the  bowels  of  infants  suffering 
from  the  disease  also  contain  the  infective  material. 

12.  Milk,  though  a  much  more  likely  channel  for  conveyance  of  tubercle  than 
meat,  is  only  or  chiefly  virulent  when  the  udder  is  the  seat  of  tuberculous  lesions. 
The  consumption  of  such  milk  is  only  dangerous  when  it  contains  a  great  number  of 
bacilli  and  is  ingested  in  considerable  quantity.     Practically,  the  danger  from  using 
raw  milk  only  exists  for  those  persons  who  use  it  as  their  sole  or  principal  food,  e.g. , 
young  children.     All  danger  is  avoided  by  boiling  or  pasteurising  the  milk. 

At  the  same  time  there  is  an  increasing  amount  of  evidence  forthcoming  at  the 
present  time  which  goes  to  prove  that  milk  is  not  infrequently  tainted  with  tubercle 
(see  pp.  204-206).  The  tuberculin  test  should  be  applied  to  all  milch  cows,  and  the 
infected  ones  isolated  from  the  herd.  They  need  not  necessarily  be  slaughtered. 
Milk  supplies  should  be  more  strictly  inspected. 

13.  There  are  several  methods  by  which  meat  infection  can  he  prevented.     In  the 
first  place,  herds  should  be  kept  healthy,  and  tubercular  animals  isolated.    Cowsheds 
and  byres  should  be  under  sanitary  supervision,  especially  as  regards  overcrowding, 
dampness,    lack    of   light,   and    uncleanliness.      Public  slaughter-houses  under  a 
Sanitary  Authority  would  undoubtedly  be  advantageous.     Meat  inspection  should 
also  be  more  strictly  attended  to;  efficient  cooking,  and  avoidance  of  "roll"  meat 
which  has  not  been  thoroughly  cooked  in  the  middle,  are  also  wise  measures  to  adopt. 

14.  Consumptive  patients  may  diminish  their  disease. — Dr  Arthur  Ransome*  has 
laid  down  five  axioms  of  hygiene  for  phthisical  patients  which,  if  followed,  would 
materially  improve  the  condition  of  such  persons.     At  Davos,  St  Moritz,  Nordrach, 
Nordrach-on-Mendip,  and  many  other  places  where  they  have  been  practised,  the 
beneficial  change  has  been  in  many  cases  extraordinary. 

(1)  Abundance  of  light,  nutritious,  easily  digested  food,  which  must  comprise 

a  large  allowance  of  fat ;  small  meals,  but  frequent. 

(2)  An    almost    entirely  open-air    life,  with  as  much   sunshine  as   can  be 

obtained. 

Suitable  clothing,  mostly  wool. 
Cleanliness,  and  bracing,  cold-water  treatment. 
(5)  Mild  but  regular  exercise. 

15.  Consumptive  patients  may  also  assist  in  preventing  the  spread  of  the  disease. — In 
the  first  place,  they  should  follow  the  hygienic  directions  just  mentioned,  because  the 
fulfilment  of  such  conditions  will  materially  lessen  the  contagiousness  of  the  disease. 
Next,  the  expectoration  must  never  be  allowed  to  get  dry.     A  spitting-cup  containing 
a  little  disinfectant  solution  (one  teaspoonful  of  strong  carbolic  acid  to  two  table- 
spoonfuls  of  water)  should  always  be  used,  or  the  expectoration  received  into  paper 
handkerchiefs  which  can  be  burnt.     Spoons,  forks,  cups,  and  all  such  articles  should 
be  thoroughly  cleaned  before  being  used  by  other  persons.     The  patient  should  not 
sleep  in  company  with  another,  and  should  occupy,  if  possible,  a  separate  bedroom. 
Isolation  hospitals  for  consumptives,  as   for   patients   suffering  from  the  ordinary 
infectious  diseases,  are  now  being  established. 

16.  House  influence  has  some  effect,  both  directly  and  indirectly,  upon  tubercular 
diseases.     Damp  soils,  darkness,  and  small  cubic  space  in  the  dwelling-house  exert  a 
very  prejudicial  effect  upon  tubercular  patients.     Sir  Richard  Thorne  Throne  t  has 

*  Arthur  Ransome,  M.D.,  F.R.S.,  Treatment  of  Phthisis. 
t  Practitioner,  vol.  xlvi. 


(3) 

(4) 


PREVENTION  OF  TUBERCULOSIS  357 

of  bacilli  of  which  the  chief  is  the  B.  pseudo-tuberculosis  of  Pfeiffer.* 
Other  workers  have  described  very  similar  organisms. 

described  the  favourable  house  for  such  persons  as  one  built  upon  a  soil  which  is  dry 
naturally  or  freed  by  artificial  means  from  the  injurious  influences  of  dampness  and 
of  the  fluctuations  of  the  ground  water.  The  house  itself  should  be  so  constructed  as 
to  be  protected  against  dampness  of  site,  foundations,  and  walls.  Upon  at  least  two 
opposite  sides  of  the  dwelling-house  there  should  be  enough  open  space  to  secure 
ample  movement  of  air  about  it,  and  free  exposure  to  sunlight.  Lastly,  it  should  be 
possible  to  have  free  movement  of  air  by  day  and  night  through  all  habitable  rooms 
of  the  house.  It  is  clear  many  inhabited  houses  could  not  stand  these  tests ;  but 
effort  should  be  made  to  approach  as  near  to  such  a  standard  as  possible. 

17.  Tubercle-infected  Houses. — Many  authorities  have  demonstrated  the  fact  that 
dust  in  houses  may  contain  the  tubercle  bacillus,  and  that  thus,  presumably,  persons 
may  become  infected.     In  1904,  Klein  found  living  tubercle  bacilli  in  the  sweepings 
of  the  floors  of  public-houses,  and  some  fifteen  years  ago  Cornet  published  the 
result  of  his  investigations  into  the  infectivity  of  the  dust  found  in  the  dwellings 
of  consumptives  in  Berlin,  and  some  work  of  a  similar  character  has  been  done  in 
England  by  Coates.  * 

These  investigations  consisted  of  bacteriological  examinations  of  dust  collected  in 
houses  of  three  types  : — 

(a)  Dirty  houses  in  which  a  consumptive  patient  is  living  who  takes  no  precau- 
tions to  dispose  of  his  expectoration,  but  spits  freely  upon  the  floor  and  into  his 
pocket  handkerchief.  In  6 6  '6  per  cent,  of  these  houses  virulent  tubercle  bacilli  were 
found  showing  the  large  amount  of  dangerous  infective  material  present  in  an  in- 
fected house. 

(6)  CJean  houses  in  which  a  patient  is  living  who  is  not  sufficiently  careful  as  to 
the  disposal  of  his  sputa.  In  50  per  cent,  of  these  instances  the  bacillus  was  found. 
It  is  evident  that  ordinary  household  cleanliness  alone  is  insufficient  to  prevent  the 
accumulation  of  infective  material  in  rooms  occupied  by  a  consumptive. 

(c)  Very  dirty  houses  in  which  there  had  been  no  case  of  consumption  for  some 
years.  In  this  class  of  house  no  tubercle  bacilli  were  present,  showing  that  virulent 
dust  found  in  classes  1  and  2  must  have  been  due  to  the  presence  of  the  consumptive 
patient. 

Taking  the  first  two  classes,  the  average  of  houses  infected  was  61  per  cent. 
Cornet's  similar  work  resulted  in  finding  71  per  cent,  tubercle  infected. 

It  was  ascertained  that  the  dust  nearer  the  floor  than  the  ceiling  possessed  the 
greatest  virulency.  It  was  also  shown  that  the  infective  dust  was  most  virulent  in 
cases  where  the  access  of  sunlight  and  free  circulation  of  air  was  prevented,  while, 
conversely,  the  beneficial  effect  of  light  and  air  was  demonstrated  even  in  the  dirtiest 
houses.  Instances  were  given  of  the  dangers  attaching  to  infected  rooms,  and  the 
risk  to  healthy  occupants  arid  their  successors.  According  to  Koch,  "it  is  the  over- 
crowded dwellings  of  the  poor  that  we  have  to  regard  as  the  real  breeding-places  of 
tuberculosis. " 

18.  Sunlight  and  fresh  air  are  the  greatest  enemies  to  infection. 

19.  Disinfection  is  necessary  after  death  from  phthisis,  and  should  be  as  complete 
as  after  any  other  infective  disease.     Compulsory  notification   of  fatal  cases  and 
compulsory  disinfection  have  been  officially  ordered  by  the  Prussian  Government. 
In  this  country,  also,  absolute  disinfection  should  always   be  insisted  upon  after 
phthisis.       Walls,  floors,   carpets,   curtains,   etc.,   should   be   strictly    disinfected. 
Spraying  with  1-100  solution  of  chloride  of  lime,  or  other  similar  disinfectant,   is 
the  best  method  (see  p.  444). 

*  The  pathology  and  etiology  of  pseudo-tuberculosis  is  fully  treated  of  by 
Klein  in  the  Supplement  to  the  Twenty-Ninth  Annual  Report  of  the  Local  Government 
Board,  1899-1900,  pp.  355-384.  See  also  Annales  de  rinstitut  Pasteur,  1894,  No.  4, 
and  Jour,  of  Path,  and  Bact.,  1898,  pp.  160-181  (Muir). 

*  Trans.  Brit.  Gong,  on  Tuberculosis,  1901,  vol.  ii.,  pp.  88-101. 


358     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

The  B.  pseudo-tuberculosis  (Pfeiffer)  resembles  B.  coll,  and  occurs  as  short,  small 
bacilli,  cylindrical,  and  with  round  ends.  Its  manner  of  grouping  is  singly,  or  in 
couples  or  chains ;  sometimes  filamentous  forms  and  long  chains  occur  in 
bouillon  culture.  It  stains  by  alkaline  Loffler's  methyl-blue,  and  also  by  Gram's 
method  (Klein).  It  is  non-motile.  In  bouillon,  in  twenty-four  hours  a  well-marked 
granular  cloudiness  appears,  and  small  flocculi  float  through  the  liquid.  Imperfect 
pellicle  after  several  days'  growth.  No  general  turbidity.  On  gelatine  the  growth 
resembles  B.  coli,  but  the  colonies  are  more  circumscribed  and  granular,  and  later, 
they  become  tuberculated.  Growth  is  slow,  and  the  colonies  become  more  opaque, 
whiter,  and  less  spread  out  than  B.  coli.  No  gas  is  formed  in  gelatine  shake  cultures. 
There  is  no  liquefaction  of  gelatine.  On  agar  minute  grey-white  flat  colonies  appear. 
Stroke  and  stab  cultures  are  similar  to'  B.  coli,  but  not  so  luxuriant.  There  is 
limited  growth  on  potato,  which  forms  a  thin  layer  with  crenated  thicker  margin  of 
a  whitish-yellow  colour.  It  grows  well  in  milk,  but  leaves  it  unaltered.  Patho- 
genesis— Guinea-pigs  inoculated  subcutaneously  with  a  small  quantity  of  culture  die 
in  a  few  weeks.  Their  organs  are  found  to  be  studded  with  yellow-white  nodules 
containing  the  bacillus  in  pure  culture.  These  nodules  develop  more  rapidly  than 
true  tuberculosis,  but  do  not  contain  any  giant  cells.  If  fed  with  food  contaminated 
with  this  organism,  similar  nodules  develop  in  the  walls  of  the  intestine  and  mesenteric 
glands.  Klein  believes  that :  *'  The  presence  of  the  B.  pseudo-tuberculosis  in  milk  may 
probably  play  a  part  in  causing  pseudo-tuberculous  disease  in  the  human  subject. " 

Klein  found  this  bacillus  present  in  2  out  of  5  samples  of  London 
milk,*  and  in  8  out  of  100  samples  of  country  milk  delivered  in 
London.f  Delepine  found  that  out  of  450  samples  of  milk,  lesions 
produced  by  pseudo-tubercle  bacilli  were  met  with  four  times.  It 
seems  not  unlikely  that  this  group  of  bacilli  includes  several  varieties 
bearing  a  close  general  resemblance  to  each  other,  but  possessing 
slightly  different  properties.  They  gain  access  to  milk  in  all 
probability  by  some  accidental  contamination.  The  milk  itself 
remains  unaltered  in  appearance,  though  it  becomes  alkaline.  As 
regards  differential  diagnosis,  it  may  be  said  that  the  pseudo-tubercle 
bacillus  is  not  acid-fast,  nor  is  it  similar  to  B.  tuberculosis  in  morpho- 
logical or  cultural  characters.  The  pathological  changes  set  up  by 
it,  and  which  form  its  chief  claim  to  be  considered  as  in  any  way 
related  to  tuberculosis,  differ  from  that  disease  in  showing  an  absence 
of  giant  cells  in  the  nodules,  absence  of  the  true  tubercle  bacilli, 
copious  presence  of  the  pseudo-tubercle  bacilli,  and  a  more  rapid 
development  of  disease. 


ACID-FAST  BACILLI  ALLIED  TO  THE  TUBERCLE  BACILLUS 

We  may  here  suitably  consider  the  group  of  organisms 
morphologically  and  tinctorially  similar  to  the  bacillus  tuberculosis. 
This  group  is  known  as  that  of  the  acid-fast  bacilli,  on  account  of 
the  fact  that  in  staining  by  the  Ziehl-Neelsen  method  (see  p.  459) 
these  organisms  possess,  like  the  tubercle  bacillus,  the  power  of 

*  Report  of  Local  Government  Board,  1899-1900,  p.  360,  and  1900-1901,  p.  3.°,2. 
t  Jour,  of  Hygiene,  1901,  vol.  i.,  p.  83. 


ACID-FAST  ORGANISMS  359 

resisting  decolorisation  by  the  acid  following  the  red  stain.*  In 
England  such  bacilli  are  termed  acid-fast,  in  Germany  saurefcste, 
and  in  France  acidopliile.  The  group  is  one  of  great  importance, 
partly  on  account  of  the  ease  with  which  its  members  may  be 
mistaken  for  the  "  true  "  tubercle  bacillus,  and  partly  on  account  of 
the  relationship  which  appears  to  exist  between  them  and  the 
tubercle  bacillus.  Some  bacteriologists  hold  that  possibly  these 
acid-fast  bacilli  represent  a  saprophytic  stage  in  the  life-history  of 
the  true  tubercle  bacillus. 

In  his  description  of  the  tubercle  bacillus,  Koch  foretold  the 
probability  of  other  acid-fast  organisms  being  discovered,  and  some 
fourteen  years  after,  in  1896,  Koch  and  Petri  actually  demonstrated 
the  occurrence  of  such  bacilli  in  the  butter  and  milk  of  Berlin.  In 
the  years  immediately  following,  Eabinowitsch,  Korn,  Coggi,  Tobler, 
and  others,  found  further  organisms  of  this  nature  in  such  articles 
of  food.  In  1898  Moeller  showed  that  these  acid-fast  bacilli 
occurred  naturally  in  animals  and  plants.  Dust,  grass,  hay,  manure, 
and  similar  substances  yielded  them,  and  now  it  is  known  that  a 
considerable  family  of  these  bacteria  exists.  It  should,  however,  be 
understood  that  the  group  is  provisional  only.  Further  knowledge 
may  reveal  facts  which  would  considerably  modify  present  views. 

Classification  of  Acid  -  fast  Bacilli.— These  bacilli  may  be 
divided  provisionally  and  for  convenience  into  four  chief  sub- 
divisions : — 

(a)  The  acid-fast  bacilli  of  other  diseases  or  conditions  affecting 
man  (e.g.,  B.  leprce,  B.  smegmatis,  B.  of  syphilis  of  Lustgarten,  etc.). 
Other   non-tubercular   acid-fast   bacilli   have   been   found   in    lung 
gangrene  (Frankel),  in   the   nasal  cavities  (Karlinski),   in   excreta, 
and  in  certain  chronic  eye  diseases,  etc. 

(b)  The   acid-fast   bacilli   occurring   in   other   animals   (e.g.,   B. 
tuberculosis  avium  of  Maffucci;  the  B.  tuberculosis  piscium  of  Dubard, 
Bataillon,  Terre ;  B.  tuberculosis  ranicola  of  Lubarsch ;  B.  tuberculosis 
anguicola  of  Moeller,  etc.). 

(c)  The  acid -fast  bacteria  of   butter   and   milk   (e.g.,  B.  laticola 
planus,perrugosum,  Friburgense,  etc.),  of  Petri,  Moeller,  Eabinowitsch, 
Binot,  Markl,  Coggi,  Tobler  (Nos.  i.-v.),  Grassberger,  and  Korn  (Nos. 
i.  and  ii.). 

(d)  The  acid-fast  bacilli  of  grass  (e.g.,  B.  pJilei  or  Timothy  lacillus, 
and    Grass   bacillus,  No.  ii.,  of  Moeller),  the  "manure  bacillus"  of 
Moeller,  the  urine  bacillus  of  Marpmann. 

All  these  various  organisms  are  morphologically  and  in  staining 

*  Acid-fastness  is  due,  in  all  probability,  not  to  fat  in  the  bacillus,  but  to  a 
substance  of  the  nature  of  wax,  which  can  be  extracted  by  acid-alcohol,  ether,  or 
other  wax  solvents.  For  a  discussion  of  this  subject,  see  Trans.  British  Congress  of 
TvbtreuMi,  1901,  vol.  iii.,  pp.  498-502  (Bulloch). 


360    TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

properties  allied  to  the  B.  tuberculosis.  The  chief  characters  of  most 
of  them  are  referred  to  under  Tuberculosis,  Leprosy,  etc.,  but  it  will 
be  necessary  to  describe  briefly  the  character  of  others. 

(a)  Acid-fast  Bacilli  of  Human  Origin. —The  leprosy  bacillus  will 
be  described  subsequently  (see  p.  398). 

The  smegma  bacillus  was  first  discovered  by  Tavel  and  Alvarez, 
in  1885,  in  the  normal  preputial  smegma,  and  also  in  the  secretion  of 
the  outer  skin,  particularly  where  a  collection  of  epithelium  may 
occur,  as  in  the  fold  of  the  groin,  between  the  toes,  etc.  The 
discovery  of  the  bacillus  was  incidentally  made  in  investigating  an 
observation  of  Lustgarten,  in  1884,  on  the  syphilis  bacillus. 
Morphologically  and  tinctorially,  the  smegma  bacillus  closely 
resembles  the  syphilis  bacillus  of  Lustgarten.  This  fact  discounted 
the  importance  attached  to  Lustgarten's  discovery,  and  subsequent 
investigations  show  that  Lustgarten's  bacillus  has  not  been  found  in 
sufficient  numbers,  or  with  sufficient  constancy,  in  the  syphilitic  tissue. 

The  smegma  bacillus,  according  to  Tavel  and  Alvarez,  is  morpho- 
logically exceedingly  like  the  tubercle  bacillus,  and  can  be  stained 
by  the  same  methods.  Inoculation  experiments  on  animals  were 
without  result,  nor  were  the  authors  able  to  obtain  a  pure  culture. 
Laser  and  Czaplewski  have  cultivated  (the  former  from  the  secretion  of 
syphilitic  affections,  the  latter  from  gonorrhoeal  pus)  micro-organisms 
resistant  to  acids,  similar  to  diphtheria  bacilli,  which  have 
been  declared  by  both  authors  to  be  identical  with  the  smegma 
bacillus.  Frankel  only  calls  those  micro-organisms  smegma  bacillus 
which  first  attracted  attention  by  their  great  resemblance  to  tubercle 
bacilli.  This  resemblance  is  wanting  in  the  cultures  of  Czaplewski 
and  Laser,  and  in  his  own  cultures,  which  he  described  later.  In 
form  and  other  characters  they  are  much  more  like  a  pseudo- 
diphtheritic  bacillus.  Moeller  agrees  with  Frankel.  Moeller  was 
not  able  to  get  a  pathogenic  effect  in  guinea-pigs  either  with  the 
diphtheroid  bacillus  cultivated  in  pure  culture  from  smegma,  or  with 
the  genuine  smegma  bacillus  containing  cutaneous  secretion  in 
abundance.  In  this  way  the  smegma  bacillus  differs  from  other 
acid-fast  bacilli.  He  found  human  serum  the  best  culture  medium 
for  smegma  bacillus.  The  morphology  differs  according  to  media, 
especially  in  milk  cultures.  Moeller  found  the  bacillus  to  be 
absolutely  acid-fast  and  alcohol-fast,  and  this  property  is  not  much 
diminished  by  age  or  media.  The  organism  is  strongly  aerobic,  and 
grows  slowly.  On  glycerine  agar  at  37°  C.  dull  grey-white  scales 
of  growth  occur,  and  on  potato  dull  white-grey  colonies.  Growth  is 
rapid  in  milk,  and  the  milk  is  not  coagulated. 

With  respect  to  differential  diagnosis,  especially  in  reference  to 
urogenital  tuberculosis,  the  smegma  bacillus  is  undoubtedly  of  great 
importance. 


Acid-fast  bacillus  from  butter  of  Berlin 

(Petri-Rabinowitsch). 

Flask  culture  on  glycerine-agar — 3  months  at  22°  C. 
Single  colony — actual  size. 


Acid-fast  bacillus  from  milk  of  Belzig 

(A.   Moeller). 

Flask  culture  on  glycerine-agar — 3  months  at  22°  C. 
Single  colony — actual  size. 


To  face  page  360. 


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ACID-FAST  BACILLI  IN  BUTTER  AND  MILK  361 

Neufeld  and  others  have  obtained  acid-fast  organisms  from 
smegma,  and  Neufeld  concludes  that  some  of  these  may  be  described 
as  similar  to  B.  diphtherias  and  others  to  the  tubercle  bacillus. 

(b)  Acid-fast  Tubercle  Bacilli  in  Animals.— The  members   of 
this  group  are  provisionally  assumed  to  be  related  to  the  tubercle 
bacillus.     Eeference  is  made  to  these  organisms  under  Tuberculosis 
(see  p.  349). 

(c)  Acid-fast  Bacilli  in  Butter  and  Milk.— Several  years  after 
Koch's  discovery  of  the  tubercle  bacillus,  species  of  bacteria  were 
found  possessing  acid-fast  properties,  but  it  was  not  until  1896  that 
Koch  and  Petri  isolated  such  organisms  from  the  milk  and  butter  of 
Berlin,  and  in  the  following  year  Lydia  Rabinowitsch  carried  out  her 
research  on  the  same  subject.     In  1899,  Korn  discovered  two  bacilli 
in  the  butter  of  Freiburg,  Binot  a  bacillus  in  the  butter  of  Paris, 
and  Coggi  a  bacillus  in  the  butter  of  Milan,  all  four  of  which  were 
acid-fast.     In  the  same  year  G-rassberger  published  a  statement  upon 
acid-fast  organisms  occurring  in  butter  and    margarine.     In  1900, 
Beck  and  Santori  met  with  similar  organisms  in  milk;  and  in  1901, 
Maria  Tobler,  Markl,  and  Moeller  and  Jong  isolated  acid-fast  bacilli 
from  both  butter  and  milk.     All  these  organisms  were  bacilli,  but 
they   showed   much   variation   in   form    and    polymorphism,   some 
appearing  to  be  like   B.  diphtheria,   and   others   like  actinomyces. 
The  staining  properties  were,  in  all  cases,  those  of  the  true  bacillus 
of  Koch,  except  that  the  power  of  resistance  to  decoloration  by  acid 
was  rather  less.*     Swithinbank  has  cultured  many  of  these  organ- 
isms upon  different  media,  and  has  found  them  to  show  various 
modifications  in  form,  chromogenicity,  vitality,  polymorphism,  etc. 
By  a  series  of  cultures,  he  showed  the  various  characters  of  ten  of 
these  acid-fast  species  compared  with  the  human  and  bovine  tubercle 
bacillus,  all  the  cultures  having  been  grown  on  the  same  media,  for 
approximately  the  same  length  of  time,  under  precisely  the  same  con- 
ditions.   The  cultures  were  in  each  case  composed  of  one  colony  only, 
and  admirably  revealed  the  differences  between  the  species.     These 
acid-fast  bacilli  live  and  develop  on  all  ordinary  media  at  room  tem- 
perature and  blood-heat,  preferably  under  aerobic  conditions.     They 
do  not  form  indol  or  liquefy  gelatine,  nor  do  they  possess  much  patho- 
genic action.-f 

The  Butter  Bacillus  of  Petri- Rabinowitsch. — Morphologically,  this 
organism  is  like  the  ordinary  tubercle  bacillus,  though  somewhat 
shorter  and  thicker.  It  stains  in  the  same  manner,  but  grows  readily 
at  room  temperature  and  rapidly  at  blood-heat.  It  is  non-motile. 

*  Report  of  Medical  Officer  of  Local  Government  Board,  1900-1,  pp.  331-3. 

t  See  Bacteriology  of  Milk  (Swithinbank  and  Newman) ;  in  The  Journal  of  State 
Medicine,  1903-4,  will  be  found  a  useful  summary  of  present  knowledge  of  acid-fast 
bacteria,  by  A.  C.  Coles  ;  Potet's  work  should  also  be  consulted. 


362     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

The  cultures  appear  as  moist,  thick,  creamy,  wrinkled  layers  of  growth 
on  the  surface  of  the  medium.  The  bacillus  renders  broth  turbid  and 
acid.  Indol  is  also  produced,  and  a  disagreeable  odour.  The  growth 
is  slow  on  gelatine,  occurring  as  small  granular  colonies.  It  is  non- 
liquefying.  On  glycerine  agar,  growth  is  abundant,  rapid,  and  char- 
acteristic. It  occurs  as  a  creamy  film — of  a  light  golden  colour — 
moist,  thick,  and  much  wrinkled  (see  Plate).  It  possesses  a 
glistening  appearance,  and  an  unpleasant  odour  These  characters 
disappear  after  much  sub-culturing.  Milk  is  not  coagulated.  A  dull 
dry  growth  generally  occurs  on  potato.  The  organism  possesses  less 
virulence  when  inoculated  in  pure  culture.  But  when  inoculated 
with,  or  without,  butter,  it  has  clearly  defined  effects.  Giant  cells, 
nests  of  epithelioid  cells,  and  typical  tuberculous  cassation  are, 
according  to  Eabinowitsch,  never  to  be  found  in  the  foci  of  disease 
set  up  by  this  bacillus.  None  of  the  animals  injected  with  this 
bacillus  reacted  to  tuberculin.  The  intra-peritoneal  injection  of  pure 
cultures  often  produces  a  formation  of  nodules  in  the  abdominal 
organs  which  frequently  heal.  If,  however,  the  animals  are  killed  in 
three  or  four  weeks,  the  following  characteristics  are  found,  namely, 
slightly  distended  abdomen,  more  or  less  severe  peritonitis,  nodules 
on  mesentery  and  beneath  the  intestinal  serosa,  mesenteric  glands 
enlarged,  and  liver,  spleen,  and  kidneys  showing  small  nodules  with 
yellowish  exudation.  When  the  butter  itself  containing  the  organisms 
is  used,  a  fatal  result  often  follows  the  injection  after  three  to  fifteen 
days.  Similar  changes  to  the  above  have  occurred,  but  of  a  more 
intense  degree.  Kabinowitsch  found  rabbits  insusceptible  in  contrast 
to  guinea-pigs.  It  is  not  known  whether  this  bacillus  is  in  any 
degree  pathogenic  for  man.  But  probably  such  is  not  the  case.  It 
appears  to  be  widely  distributed  in  nature,  as  60  per  cent,  of  butter 
samples  in  Berlin  were  found  to  contain  it.  The  only  satisfactory 
way  to  differentiate  this  bacillus  from  the  tubercle  bacillus  is  by 
inoculation  of  animals. 

Moeller  isolated  a  somewhat  similar  organism  from  milk, 
which  is  generally  known  as  Moellcr's  Milch  Bacillus.  It  was  found 
in  pasteurised  milk  at  Belzig,  and  is  almost  identical  in  morphology 
to  the  tubercle  bacillus.  It  is  acid-fast,  non-motile,  and  grows  at 
room  temperature  as  well  as  blood-heat.  Broth  becomes  but  little 
turbid,  and  there  is  no  deposit.  Surface  membrane  of  fatty  aspect 
and  amber  colour,  adherent  to  tube  walls,  is  sometimes  formed.  On 
gelatine  plates  and  tubes  a  white  wrinkled  culture  of  creamy  nature 
occurs,  and  on  glycerine  agar,  after  about  three  weeks,  the  growth  is 
white,  uniform,  and  of  a  creamy  nature,  though  at  times  slightly 
wrinkled,  and  dry.  In  old  cultures  it  is  dry,  or  glazed,  and  of  a 
yellowish  colour,  which  later  turns  to  a  reddish  tint,  the  culture 
itself  becoming  of  a  wrinkled  appearance.  Frequently  the  raised 


PLATE  26. 


Acid-fast  bacillus  from  butter  of  Friburg 

(Bacillus  Friburgensis — Korn  I.) 

Flask  culture  on  glycerine-agar — 3  months  at  22°  C. 

Single  colony — actual  size. 


Acid-fast  bacillus  from  butter  of  Friburg 

(Korn  II.) 

Flask  culture  on  glycerine-agar — 3  months  at  22°  C. 
Single  colony — actual  size. 


To  face  faye  362* 


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ACID-FAST  BACILLI  IN  BUTTER  AND  MILK  363 

"  granular  "  centre  is  surrounded  by  a  light  blue  zone,  but  slightly 
raised  above  the  medium  (see  Plate) ;  on  glycerine  potato,  there  is 
at  first  a  white  creamy  growth  very  little  raised  above  the  surface  of 
the  medium,  but  as  it  grows  older  the  culture  becomes  wrinkled  ^and 
of  a  deep  yellowish  tint,  almost  red.  The  bacillus  grows  quickly  and 
luxuriantly  in  milk,  forming  an  ochre-yellow  ring  round  the  surface 
edge  of  the  medium.  The  organism  was  found  to  produce  nodules  in 
the  organs  of  inoculated  animals. 

Korn  isolated  two  acid-fast  bacilli  from  Friburg  butter.  B.  fri- 
buryensis,  No.  1,  varies  in  morphology  under  different  circumstances. 
In  preparations  made  from  the  organs  of  an  inoculated  animal,  the 
bacilli  resemble  in  shape  and  size  the  B.  tuberculosis  of  Koch.  In 
bouillon,  the  shape  very  much  resembles  the  B.  coli,  but  is  a  little 
longer  and  slightly  curved.  On  agar  the  bacilli  are  slightly  thinner. 
In  old  cultures  upon  agar  and  serum,  they  assume  the  aspect  of 
"  Coccothrix."  Upon  potato  they  appear  under  form  of  cocci,  diplo- 
cocci,  and  specially  of  short,  stout  bacilli  slightly  curved.  Upon 
cooked  beetroot  at  the  end  of  three  or  four  days  the  organisms 
resemble  staphylococci.  They  are  shorter  when  grown  at  ordinary 
temperature  than  when  grown  at  37°  C.  In  culture  media 
the  older  growths  generally  exhibit  some  orange  or  red  coloration, 
though  on  almost  all  media  the  growth  is  at  first  white  and 
non-wrinkled.  Upon  glycerine-agar  a  thick,  white,  brilliant  growth 
occurs  with  deposit  in  the  water  of  condensation,  which  is  soon 
covered  with  a  veil  adherent  to  the  walls  of  the  tube.  Later 
on  the  growth  becomes  slightly  folded.  At  the  age  of  about  three 
weeks  the  culture  is  creamy,  presenting  a  few,  light  folds,  and 
of  yellow-orange  colour.  In  older  cultures  the  growth  is  very 
abundant,  raised  considerably  above  the  surface,  and  irregularly 
folded  and  convoluted,  the  colour  varying  in  depth  from  light  to 
dark  orange  or  even  red  brick  (see  Plate).  Milk  is  not  coagu- 
lated, but  at  the  end  of  three  weeks  possesses  a  yellowish-brown 
colour.  Subcutaneous  or  intraperitoneal  injection  of  pure  cultures 
produce  only  an  abscess  at  site.  If  injected  with  butter  itself  in 
white  mice,  granulations  are  produced  in  thoracic  and  abdominal 
viscera,  showing  no  giant  cells,  but  commencing  caseation. 

B.  friburgensis,  No.  2,  consists  of  a  small  rod  two  or  three  times 
longer  than  broad,  often  irregular  and  sometimes  clubbed.  It  is 
much  less  sensitive  to  decolorisation  by  acid  than  No.  1 ;  grows 
feebly  on  ordinary  laboratory  media,  with  the  exception  of  glycerine 
agar,  in  which  growth  appears  in  twenty-four  hours,  and  eventually 
becomes  abundant.  The  culture  is  creamy,  glistening,  and  a 
yellowish  colour  (see  Plate).  Milk  turns  of  a  dirty  red  colour  in 
about  three  weeks  time. 

Markl,  Tobler,  Coggi,  Binot,  and  Grassberger  have  also  isolated 


364     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

acid-fast  bacilli  from  butter.  Brief  reference  may  be  made  to  the 
organisms  discovered  by  the  two  last-named  workers. 

Binot's  Butter  Bacillus  is  in  morphological  and  tinctorial  char- 
acters similar  to  the  tubercle  bacillus.  It  differs  in  cultural  features. 
Binot  describes  it  as  producing  a  thick,  viscous,  creamy  layer  in 
broth,  from  which  "  stalactites  "  grow  down  into  the  liquid.  It  grows 
on  gelatine  (which  it  does  not  liquefy),  producing  thin  creamy  irregu- 
lar surface  colonies.  On  glycerine-agar  plates  and  tubes  white  colonies 
appear,  becoming  straw-coloured,  and  finally  orange.  They  may 
attain  diameter  of  two-franc  piece  or  larger;  and  have  a  bright 
glistening  surface,  and  are  very  adherent  to  medium.  On  the  surface 
they  soon  become  wrinkled  and  irregular,  and  have  scalloped  edges. 
Chromogenic  characters  are  more  marked  if  the  growth  is  exposed  to 
air  and  light  (see  Plate).  On  potato,  a  scanty  growth  occurs,  which 
is  at  first  moist,  and  of  a  clear  yellow  colour.  On  glycerine  potato 
an  abundant  homogeneous  growth  occurs,  of  an  opaque  yellow  colour, 
turning  to  orange.  Irregular  nodosities  appear  on  the  colony.  The 
bacillus  produces  tuberculous-like  changes  in  animals  in  which  it  is 
inoculated. 

The  Butter  Bacillus  of  G-rasslerger  is  very  similar  to  the  other 
members  of  this  group,  except  that  it  produces,  especially  on  gelatine, 
but  also  on  glycerine  agar,  a  dry,  much-wrinkled  growth  not  unlike 
some  forms  of  mould,  and  of  a  deep  rose  colour.  There  is  no  liquefac- 
tion. The  wrinkles  in  the  large  colonies  appear  as  characteristic 
markings  (see  Plate).  Milk  is  coloured  throughout  by  the  organism, 
but  not  coagulated.  There  is  also  a  surface  growth  and  deposit,  both 
rose  coloured.  On  potato  the  growth  is  similar  to  tubercle  bacillus, 
but  of  a  deep  rose  tint. 

(d)  Acid-fast  Bacilli  in  Grass,  Hay,  and  Manure. — This  second 
group  of  acid-fast  bacilli  associated  with  milk  (marked  (d)  in  the 
classification  above)  is  often  designated  as  that  of  the  grass  bacilli. 
They  were  first  cultured  on  Timothy  grass  (Phleum  pratense),  which 
is  much  valued  for  feeding  cattle.*  Since  then,  however,  this  grass 
bacillus  has  been  found  in  various  places,  and  it  or  its  allies  have 
been  isolated  from  cattle  fodder,  hay,  hay-dust,  manure,  milk  and 
its  derivatives.  Morphologically,  this  bacillus  (B.  phlei)  is  similar  to 
the  tubercle  bacillus,  slender  and  slightly  curved.  It  contains 
highly-stained  granules  and  oval,  clear  spaces ;  often  grows  in  threads ; 
and  is  branched,  and  sometimes  has  clubbed  swellings  at  one  end. 
It  is  acid-fast  in  staining,  and  grows  best  at  incubation  temperatures 
on  the  ordinary  culture  media.  The  colonies  become  visible  in  thirty- 


*  Cat's  tail  or  Timothy  grass  (Phleum  pfatense).     Although  well  known  to 
British  grower  this  grass  is  more  extensively  cultivated  in  the  United  States,  wl 


the 
where 

it  was  introduced  from  Britain,  nearly  a  century  ago,  by  Mr  f  imotliy  Hanson,  after 
whom  it  is  named  Timothy  grass. 


Acid-fast  bacillus  from  butter  of  Paris 

(Binot). 

Flask  culture  on  glycerine-agar — 3  months  at  22°  C. 
Single  colony — actual  size. 


Acid-fast  bacillus  from  butter  of  Vienna 

(Grassberger). 

Flask  culture  on  glycerine-agar — 3  months  at  22°  C. 
Single  colony — actual   size. 


To  face  page  364 


.(lorn*!) 
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ACID-FAST  GRASS  BACILLI  365 

six  hours,  are  scale-like  and  greyish-white  or  yellow  in  colour.  It 
grows  best  on  glycerine  agar.  It  grows  slowly  in  milk,  but  does  not 
coagulate  it.  Under  certain  conditions  its  growth  in  artificial  media 
is  very  similar  to  the  tubercle  bacillus,  which,  however,  does  not  thrive 
at  room  temperature.  As  regards  pathogenic  properties,  the  grass 
bacillus  is  almost  identical  with  the  Petri-Kabinowitsch  butter 
bacillus  in  its  effects  on  guinea-pigs.  It  has  somewhat  different  effect 
on  rabbits,  producing  a  condition  difficult  to  distinguish  from  true 
tuberculosis  (Lubarsch).  Giant  cells,  epithelioid  cells,  and  caseation 
are  all  said  to  occur.  In  all  animals  injected  with  the  grass  bacillus 
a  negative  reaction  to  tuberculin  is  obtained.  Moeller  has  isolated  a 
grass  bacillus,  No.  2  (from  the  dust  of  a  hay-loft),  which  he  considers 
essentially  different  from  the  Timothy  bacillus.  The  colonies  are 
moist  and  sticky,  become  confluent,  and  are  yellow  in  colour.  It 
loses  its  acid-fast  properties  in  old  cultures.  Its  pathogenic  pro- 
perties are  most  marked  when  cultured  in  milk.  It  frequently 
shows  marked  polymorphism.  In  culture  it  is  like  the  butter  bacilli. 
Freymuth  has  shown  that  the  changes  this  organism  sets  up  in  cold- 
blooded animals  are  indistinguishable  from  true  tuberculosis.  An 
acid-fast  bacillus  similar  to  grass  bacillus,  No.  2,  has  recently  been 
isolated  by  Moeller  from  milk.  A  variety  of  the  grass  bacillus  has 
also  been  found  by  Moeller  in  the  excreta  of  animals,  and  is  therefore 
termed  the  manure  bacillus  (mist  bacillus).  This  acid-fast  bacillus 
has  been  isolated  from  the  excreta  of  cattle  and  other  animals,  and 
bears  a  morphological  and  tinctorial  resemblance  to  the  Timothy 
bacillus,  whilst  in  cultures  it  is  like  grass  bacillus,  No.  2.  On  agar 
these  organisms  grow  in  a  similar  manner,  but  bouillon  does  not 
become  turbid  with  the  growth  of  the  mist  bacillus.  It  has  certain 
pathogenic  properties.  When  injected  into  guinea-pigs,  nodules 
resulted ;  but  they  contained  few  epithelioid  cells.  The  true  radial 
arrangement  of  the  bacilli  occurs,  however.  It  is  possible  that  most  of 
the  acid-fast  bacilli  found  in  milk  and  butter  have  their  origin  in  the 
soil  or  vegetation. 

Differential  Diagnosis.— This  brief  record  of  the  acid-fast  bacilli 
is  enough  to  show  that  there  exist  a  large  number  of  bacilli,  which 
on  occasion  may  be  present  in  milk  or  milk  products,  having  char- 
acters which  ally  them  closely  to  the  tubercle  bacillus.  Moeller 
holds  that  the  primitive  form  is  the  grass  bacillus,  and  that  the 
butter  bacillus,  manure  bacillus,  etc.,  are  varieties  thereof.  The 
main  points  of  distinction  between  this  group  and  the  true  tubercle 
bacillus  are  five. 

First,  the  tubercle  bacillus  shows  a  fairly  uniform  manner  of 
growth. 

Secondly,  it  requires  incubation  temperature. 

Thirdly,  it  is  unique  with  respect  to  its  excessively  slow  growth. 


366     TUBERCULOSIS  AS  A  TYPE  OF  BACTERIAL  DISEASE 

Fourthly,  it  is  as  regards  growth  and  propagation  a  parasite. 

Fifthly,  on  inoculation  it  produces  pathological  cellular  changes 
distinct  from  the  nodular  new  growths  following  inoculation  of 
acid-fast  bacilli.  In  particular  this  is  true,  as  far  as  is  known  at 
present,  in  regard  to  the  human  organism. 

In  a  sentence,  the  acid-fast  bacilli  differ  from  the  tubercle  bacillus 
in  three  main  particulars,  viz. :  morphology  of  culture,  conditions  of 
development  (chromogenicity,  rapidity  of  growth,  range  of  tempera- 
ture within  which  they  flourish),  and  their  feebler  pathogenic  proper- 
ties. From  these  facts  it  follows  that  however  great  the  degree  of 
similarity  between  these  various  acid-fast  bacilli,  and  however  much 
it  is  possible  by  artificial  cultivation  to  modify  the  morphology  of 
the  various  forms,  there  is  sufficient  difference  to  enable  a  differ- 
ential diagnosis  to  be  made  if  all  the  biological  characters  are 
ascertained,  and  most  of  all  the  pathogenic  properties.  Hence  the 
importance  of  the  inoculation  test  being  applied  to  acid-fast  and 
tubercle-like  organisms  detected  in  milk  or  butter. 

As  a  simple  method  of  differential  diagnosis,  Moeller  suggests  that 
the  smegma,  sputum,  or  other  secretion  should  be  mixed  with  nutritive 
bouillon,  and  kept  at  about  30°  C.  If  in  two  or  three  days  there  is 
a  visible  increase  in  the  bacteria  resistant  to  acids,  it  is  certain  that 
it  is  not  the  genuine  tubercle  bacillus,  which  requires  37°  C.  Some- 
times in  sputum,  mixed  with  certain  nutritive  media,  the  tubercle 
bacillus  increases  at  incubation  temperature.  This  proliferation, 
due  in  all  probability  to  the  importation  of  globulin-like  substances 
from  the  body,  is,  however,  exceedingly  small,  and  ceases  altogether 
after,  at  the  latest,  forty-eight  hours ;  whilst  in  the  pseudo-tubercle 
bacilli  a  persistent  further  proliferation  takes  place  at  30°. 

The  pathological  differences  from  Koch's  bacillus  are  that  inocu- 
lation with  acid-fast  bacilli  gives  rise  to  110  "  giant  cells,"  no  epithe- 
lioid  cell  clusters,  and  no  tuberculous  caseation.  Nodular  lesions 
occur  suggestive  of  tubercle,  but  according  to  Potet,*  and  Abbot  and 
Gildersleive  f :  (a)  they  constitute  a  localised  lesion  only,  having  no 
tendency  to  dissemination,  metastasis,  or  progressive  destruction  of 
tissue  by  caseation ;  (b)  they  tend  to  terminate  in  suppuration  like 
ordinary  abscesses;  (c)  when  occurring  as  result  of  intravenous 
inoculation  they  appear  in  the  kidney,  rarely  in  the  lung  and 
other  organs;  and  (d)  the  form  of  granuloma  set  up  is  similar  to 
actinomyces. 

This  group  of  organisms  is  one  of  considerable  importance  to  the 
milk  bacteriologist,  and  in  all  investigations  dealing  with  the  tubercle 
bacillus,  or  with  milk  and  its  products,  it  is  essential  that  the  bacilli 
met  with  should  be  clearly  differentiated  from  the  tubercle  bacillus. 

*  Etude  sur  Us  Bacttries  elites  Acidophiles  (Potet,  Paris,  1902),  pp. 
f  The  University  of  Pennsylvania  Medical  Bulletin,  June,  1902. 


PLATE  28. 


COMPARATIVE  CULTURES  OF  ACID-FAST  BACTERIA  ON  GLYCERINE  AGAR. 

(Timothy  Grass  Bacillus,  Miller's  Grass  Bacillus,  the  Mist  Bacillus). 

(Grown  and  photographed  by  Swithinbank). 


[To  face  page  366. 


STREPTOTHRIX  GROUP  367 

Insufficient  care  has  been  taken  in  this  respect  up  to  the  present. 
Any  such  organism  found  should  be  compared  in  cultural  and 
pathogenic  properties  with  the  human  tubercle  bacillus,  the  bovine 
bacillus  of  pseudo-tuberculosis,  and  the  various  acid-fast  organisms, 
and  not  simply  accepted  on  tinctorial  properties  as  a  tubercle 
bacillus. 

Acid-fast  Streptothrix  Group. — Kecently,  considerable  attention 
has  been  given  to  the  group  of  "higher  bacteria"  known  as  the 
streptotkricece.  Seen  in  its  mature  form,  a  streptothrix  appears  to 
consist  of  a  tangled  mass  of  mycelial  threads,  some  short  bacillary 
forms,  and  spores.  The  life  cycle  is  completed  by  the  growth  or 
sprouting  of  spores  by  which  a  mycelium  is  developed.  This  branches 
off  in  various  degrees  and  directions,  and  probably  sprouts  itself,  and 
so  produces,  with  the  development  arising  from  spores,  a  fresh 
mycelial  growth.  The  mycelium  may  undergo  fragmentation,  and 
thus  bacillary  forms  occur.  Streptothrix  is  usually  readily  stained 
by  Gram's  method,  but  several  species  are  acid-fast.  Foulerton  and 
Price  Jones  found  this  to  be  characteristic  of  S.  Nocardii,  S.  caproc, 
S.  hominis  (Sabrazes),  and  S.  Eppingeri,  in  older  culture,  and  then 
only  the  mycelial  threads  and  not  the  spores.  Twenty-one  other 
species  were  not  acid-fast.*  The  germs  of  streptothrix  usually  grow 
better  in  culture  media  at  37°  C.  than  at  22°  C.,  and  better  aerobically 
than  anaerobically.  Pigment  production  occurs  in  some  forms, 
and  certain  species  liquefy  gelatine.  One  of  the  best  media  to  use 
is  maltose-peptone  agar,  but  potato,  peptone  broth,  gelatine,  or  milk 
are  also  used.  A  number  of  workers  have  now  isolated  species  of 
streptothrix  from  natural  media,  some  of  which  are  declared  to  be 
acid-fast.  Many  of  the  group  are  pathogenic  to  man  or  animals. 
S.  actinomycis  (actinomycosis),  S.  bovis  communis,  S.  madurce 
("  Madura-foot "),  and  the  disease  known  as  "  farcins  des  boeuf s  "  are 
examples.  The  organisms  found  in  lachrymal  concretions,  alveolar 
abscesses,  and  similar  conditions,  especially  in  relation  to  the  jaws, 
are  probably  frequently  streptothrical  in  origin.  These  may  gain 
access  to  the  tissues  through  carious  teeth  (by  air  or  food).  Infec- 
tion may  also  occur  through  the  tonsils,  or  cutaneously  (as  in 
Madura-foot). 

A  description  of  Streptothrix  antinomyces  will  be  found  on  a 
subsequent  page.  Here  two  forms  of  Streptothrix  isolated  by  Fouler- 
ton  from  cases  of  disease  may  be  described. 


1.  Streptothrix  luteola  (Foulerton). 

od  in   young  cultures; 


Isolated  from  a  case  of  sloughing  keratitis  in  a  girl  of  12  years. 
Staining  Characteristics, — Stains  well  by  Gram's   methi 


*  Trans,  of  the  Path.  Soc.  of  London,  1902,  vol.  liii.,  pt.  i.,  p.  Go. 


368     TUBERCULOSIS  AS  A  TYPE  OF' BACTERIAL  DISEASE 

cultures  up  to  three  months  old  show  no  acid-fast  portions  when  stained  by  the  Ziehl- 
Neelsen  method. 

Cultural  Characteristics.— Grows  freely  in  presence  of  oxygen,  very  scanty 
growth  under  anaerobic  conditions  ;  on  all  media,  except  potato,  growth  is  distinctly 
more  active  at  37°  C.  than  at  22°  C.  ;  old  cultures,  especially  those  in  peptone  beef 
broth,  have  a  faintly  feculent  odour.  On  gelatine  at  22°  C. — Streak  culture  :  obvious 
growth  is  seen  on  third  day,  at  first  of  an  opaque  white  appearance,  later  may  show 
a  very  faint  yellowish  tinge.  Growth  sinks  slowly  into  the  medium,  which  gradually 
liquefies.  Stab  culture  :  growth  occurs  along  track  of  needle  in  the  form  of  super- 
imposed colonies,  which  have  a  somewhat  flocculent  appearance.  On  peptone-maltose 
agar. — After  24  hours'  incubation  at  37°  C.  there  is  some  indication  of  growth;  at 
22°  C.  no  obvious  growth  occurs  within  this  period.  After  72  hours'  incubation  at 
37°  C.  there  is  a  fair  amount  of  growth,  and  rather  less  after  incubation  at  22°  C. 
The  growth,  at  first  of  a  faint  drab  or  whitish  tint,  after  longer  incubation,  becomes 
usually  of  a  faint  yellowish  colour.  Cultures  may  yield  a  free  formation  of  aerial 
hyphae,  giving  a  snowy  appearance  to  the  surface  of  the  growth,  or  the  surface  may 
assume  a  reticulated  appearance,  without  any  efflorescence.  (For  microscopic 
appearances,  see  Plate  29.)  On  inspissated  horse-serum.  —  Growth  is  comparatively 
scanty  on  this  medium,  appearing  after  72  hours'  incubation  at  37°  C.  At  the  end  of 
twenty-eight  days'  incubation  at  37°  C.  there  is  a  dry,  wrinkled,  drab-coloured  growth, 
which  has  sunk  slightly  into  the  medium;  no  liquefaction.  On  potato.—  Growth 
on  this  medium  is  equal  at  temperature  37°  C.  and  22°  C.  ;  at  the  end  of  48  hours' 
growth  appears  as  a  brownish  or  faintly  yellowish  stain  on  the  medium  ;  later, 
growth  usually  assumes  a  cafe  au  lait  colour;  there  is  no  pigmentation  or 
erosion  of  the  medium.  Surface  efflorescence  is  seen  only  in  cultures  incubated  at 
37°  C. ,  and  not  in  cultures  at  22°  C.  In  peptone  beef  broth.  —After  48  hours'  incu- 
bation at  37°  C.  the  appearance  of  some  filmy  growth  is  seen  at  the  bottom  of  the 
tube ;  in  older  cultures  growth  appears  as  flocculent,  more  or  less  discoid  colonies. 
No  pigmentation.  (For  microscopic  appearances,  see  Plate  29.)  In  alkaline 
litmus  milk. — Medium  at  end  of  72  hours'  incubation  at  37°  C.  is  of  a  faint  pink 
colour ;  no  coagulation.  The  pink  colour  changes  to  a  dirty  white,  and  the  milk 
clears  gradually  from  the  surface  downwards,  becoming  at  last  of  a  brown  colour. 
Growth  on  peptone  agar  is  about  equal  to  that  on  peptone-maltose  agar ;  growth  on 
glycerine  agar  (1  per  cent,  glycerine)  is  less  free  than  it  is  on  either  of  the  two  pre- 
ceding media.  Diastatic  action. — No  action  of  the  sort  is  manifested  within  fourteen 
days'  incubation  at  37°  C.  Resistance  to  heat. — Sporulating  cultures  resist  exposure 
to  moist  heat  at  70°  C.  for  20  minutes,  but  are  destroyed  by  an  exposure  to  the  same 
temperature  for  30  minutes.  Pathogenicity  for  lower  animals.— Not  found  patho- 
genic for  rabbits  (intra-venous  and  intra-peritoneal  inoculation),  for  guinea-pigs 
(intra-peritoneal  inoculation),  or  for  tame  mice  (intra-peritoneal  and  subcutaneous 
inoculation). 

2.  Streptothrix  hominis  (Foulerton). 

Isolated  from  a  case  of  pulmonary  infection  in  a  woman  (especially  from  sub- 
cutaneous abscesses). 

Staining  Characteristics. — Takes  Gram's  stain.  A  three-months'  old  culture  from 
glycerine-peptone  agar  showed  no  acid-fast  portions  when  stained  by  the  Ziehl- 
Neelsen  method. 

Cultural  Characteristics. — Growth  was  obtained  in  peptone-beef  broth  and  on 
solid  media  under  ordinary  aerobic  conditions;  no  growth  occurred  on  tubes  of 
peptone  agar  and  glycerine-peptone  agar  incubated  under  anaerobic  conditions. 
Growth  is  more  active  at  37°  C.  than  at  22°  C.  On  peptone  agar.— Growth  very 
slow  and  scanty ;  after  several  weeks'  incubation  small  whitish,  heaped-up,  dry- 
looking  colonies,  resembling  somewhat  the  growth  of  13.  tuberculosis,  are  seen.  On 
glycerine  agar  and  maltose-peptone  agar.  —The  amount  of  growth  is  very  much  the 
same  as  in  the  last  case,  and  of  much  the  same  appearance.  On  inspissated  horse- 
serum  and  inspissated  ox-serum.— Growth  is  very  scanty  ;  the  colonies  sink  slightly 
into  the  medium.  On  potato.—  No  growth  was  obtained.  In  peptone-beef  broth.— 
Small  globular  colonies  appear  in  the  depth  of  the  broth  after  about  six  days'  incu- 


PLATE  29. 


itothrix    luteola   (Foulerton).       Film    preparation    from        Streptothrix   luteola    (Foulerton).      Film    preparation    from 
jeptone-beef-broth  culture,  14  days  at  37°  C.     x  1000.  maltose  agar  culture,  6  weeks  at  37"  C.      x  1000. 


)  i 


Streptothrix  hominis  (Foulerton).     From  pus  of  small  abscess  in  chest-wall.    Stained  by  Gram's  method. 

[To  face  page  308. 


STREPTOTHRIX  HOMINTS  369 

bation  at  37°  C.  These  do  not  increase  much  in  size,  and  after  four  weeks' 
incubation  are  not  much  larger  than  a  pin's  head ;  there  is  a  tendenqy  for  the 
colonies  to  cohere  in  flocculent  masses.  The  growth  is  whitish  in  colour.  Patho- 
c/enicityfor  lower  animals. — Two  rabbits  received  intra-peritoneal  injections  of  broth 
cultures,  but  there  were  no  obvious  effects.* 

Other  acid-fast  members  of  the  pathogenic  streptothrix  group 
have  been  isolated  by  Birt  and  Leishman  from  a  case  of  pleural 
effusion ;  by  Eppinger  from  an  abscess  of  the  brain ;  by  MacCallum 
from  peritoneal  pus;  by  Nbcard  in  "farcin  du  bceuf."  These 
organisms  can,  as  a  rule,  be  differentiated  from  the  tubercle  bacillus 
by  morphological,  cultural,  and  tinctorial  properties. 

General  Note  on  Differentiation  of  Acid-fast  Organisms 

The  acid-fast  bacillus,  pseudo-tubercle  bacillus,  and  acid-fast 
streptothrix  may  all  be  found  to  resemble  the  tubercle  bacillus  in 
greater  or  less  degree.  It  has  been  suggested  that  they  are  all  of  the 
streptothrix  genus.  Whilst  they  are  all  acid-fast  they  are  not  equally 
resistant,  and  this  fact  assists  in  their  differentiation.  Broadly,  none 
of  these  forms  can  resist  decolorisation  with  25  per  cent,  sulphuric 
acid  for  more  than  sixteen  hours,  whereas  the  tubercle  bacillus  can 
withstand  decolorisation  for  seventy-two  hours  (Coles).  Hence,  if 
film  preparations  be  made  in  the  usual  way,  stained  for  seven  minutes 
with  hot  carbol  fuchsin,  and  then  decolorised  with  25  per  cent,  sul- 
phuric acid  for  sixteen  hours,  the  only  bacilli  remaining  red  are 
tubercle  bacilli.  If  this  method  fail,  cultivation  and  inoculation  tests 
must  be  applied.  The  former  by  culturing  in  broth  at  30°  C.  (shows 
growth  in  three  days  in  non-tubercle  bacilli),  the  latter  by  inoculation 
of  guinea-pigs.  And  here,  as  elsewhere,  it  is  necessary  to  observe  all 
the  characters  before  forming  a  diagnosis. 


*  An  excellent  statement  on  the  general  characteristics  and  pathogenic  action  of 
the  genus  Streptothrix  will  be  found  in  the  Trans,  of  the  Path.  Soc.  of  London, 
1902,  vol.  53,  part  i.,  pp.  56-127  (Foulerton  and  Price  Jones). 


2  A 


CHAPTEK  XI 

THE  ETIOLOGY  OF  TROPICAL  DISEASES 

Malaria :  Forms  of  Malarial  Fever,  the  Mosquito  Theory,  Prevention  of  Malaria — 
Cholera:  Methods  of  Diagnosis — Plague:  Symptoms,  Rats  and  Plague, 
Bacteriology,  Administrative  Considerations  —  Leprosy  —  Yellow  Fever  — 
Malta  Fever— Sleeping  Sickness — Beri-beri. 

IT  is  now  generally  accepted  that  the  future  prosperity  of  the  Anglo- 
Saxon  race  depends  upon  the  measure  to  which  it  is  able  to  control 
the  Tropics.  For  it  is  obvious  that  that  great  middle  band  of  the  globe 
which  we  term  the  Tropics  is  increasingly  one  of  the  most  valuable 
and  important  areas  for  colonisation  in  the  world.  Yet,  though  the 
rewards  are  great,  the  risks  and  penalties  are  also  great.  The 
coloniser  from  temperate  regions  knows  this  to  his  cost.  Malaria 
and  plague  and  cholera,  to  speak  of  no  other  tropical  diseases,  have 
made  irretrievable  claims  upon  him.  Eecently  it  has  come  to  be 
recognised  that  much  of  this  great  loss  is  preventable,  and  ought 
therefore  to  be  prevented.  The  establishment  of  Schools  of  Tropical 
Medicine  in  London,  Liverpool,  and  other  places,  and  the  practical 
means  adopted  for  preventing  cholera  epidemics  and  stamping  out 
plague  and  malaria,  are  examples  of  the  new  sense  of  responsibility 
which  is  stimulating  nations  and  governments  to  do  their  utmost 
to  bring  under  control  those  scourges  of  pestilence,  which  have  made 
the  Tropics  so  often  the  grave  of  the  white  man. 

The  channels  of  infection  in  tropical  diseases  are  various. 
Unhealthy  surroundings,  diet,  the  soil,  bad  water,  and  parasite 
hosts  (as,  for  example,  rats  in  plague  and  mosquitoes  in  malaria), 
seem  to  be  the  chief.  But  there  is  much  yet  to  be  done  in  the 
investigation  of  the  causes  of  certain  tropical  diseases. 

We  may  now  enter  upon  the  consideration  of  five  typical  diseases 
mostly  limited  to  the  Tropics :  (1)  Malaria ;  (2)  Cholera ;  (3)  Plague ; 
(4)  Leprosy ;  (5)  Yellow  Fever.  It  is  apparent  that  many  bacterial 


MALARIA  371 

diseases  are  "  cosmopolitan."  Tuberculosis,  for  example,  may  occur 
in  all  parts  of  the  world ;  so  may  pneumonia  or  typhoid.  But  the 
five  diseases  named  above  are  in  a  greater  or  lesser  degree  endennc  in 
tropical  regions.* 

1.  Malaria 

The  term  malaria  (lit.,  bad  air)  is  often  applied  rather  to  a 
group  of  fevers  than  to  one  specific  affection.  Such  fevers  have 
certain  points  in  common.  One  common  feature  is  that,  with  few 
exceptions,  the  disease  originates  in  the  blood.  A  second  feature  is 
the  elaboration  of  a  black  or  brown  pigment  from  the  haemoglobin 
present  in  the  blood  corpuscles.  And  a  third  common  character  is 
that  these  diseases  are  produced  not  by  bacteria  but  by  Ticematozoa, 
that  is  to  say,  protozoa  which  can  live  and  perform  their  functions 
in  the  blood.  The  term  "  malaria  "  should,  however,  be  reserved  for 
the  specific  disease  caused  by  the  malarial  parasite. 

For  many  years  the  group  of  diseases  represented  by  malaria 
were  designated  miasmatic,  owing  to  the  belief  that  they  were  caused 
by  some  damp  and  unhealthy  condition  of  the  soil,  from  which 
emanated  a  miasm  or  soil  ferment.  Thus  was  explained  their 
prevalence  on  and  around  marshy  tracts  of  land,  and  their  prevention 
by  land  drainage.  Whilst  these  two  latter  features  remain  true,  a 
new  interpretation  has  been  placed  upon  them. 

In  1880  Laveran  first  discovered  and  described  parasites  in 
the  blood  cells  of  malarial  patients,  and  on  further  investigation 
it  was  soon  found  that  these  assumed  many  different  forms. 
These  differences  depend  upon  the  kind  of  fever  and  the  stage  of 
fever. 

The  reasons  for  believing  that  Laveran's  bodies — though  they 
have  not  yet  been  cultivated  outside  the  human  body — are  the 
specific  cause  of  malaria  are  briefly  these : — (1)  The  parasites  found 
in  the  blood  of  malarial  patients  of  all  countries  are  the  same.  (2) 
Such  parasites  are  not  found  in  healthy  persons.  (3)  Their  develop- 
ment fully  accounts  for  the  production  of  the  melanoemia  and 
malarial  pigmentations  of  viscera  owing  to  the  melanin-forming 
property  of  the  parasite.  (4)  The  phases  in  the  development  of 
such  parasites  corresponds  with  the  clinical  course  of  malaria  (Golgi). 
(5)  Quinine,  which  cures  malaria,  kills  the  parasite.  (6)  Malaria 
can  be  conveyed  by  the  introduction  of  this  parasite  into  the  blood 

*  The  term  endemic  indicates  that  a  disease  affects  people  within  a  certain 
geographical  limit,  and  which  seems  therefore  to  arise  from  local  or  particular 
causes.  Epidemic  indicates  that  a  disease  attacks  a  large  number  of  people  at  the 
same  time  and  approximately  in  the  same  place.  Whereas  a  pandemic  is  the  same 
with  an  indefinitely  wide  distribution. 


372  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

of  man,  and  the  parasite  reappears  in  the  blood  of  the  individual  so 
inoculated.  It  is  interesting  in  this  connection  to  observe  the 
negative  results  of  the  recent  attempts  of  Koch  to  inoculate  the 
higher  apes  with  malaria  in  Batavia,  as  reported  by  the  German 
Colonial  Office.*  Laveran's  bodies  have  been  variously  classified  as 
knowledge  of  them  has  grown.  It  is  now  agreed  that  these  parasites 
belong  to  the  Sporozoa,  to  the  order  of  Hcemocytozoa,  and  to  the 
genus  of  Hcemamo&ba. 

Now  if  we  examine  a  sample  of  human  blood  from,  say,  the 
benign  tertian  form  of  malaria,  we  shall  find  not  different  parasites 
as  in  three  forms  named  below,  but  different  stages  in  the  evolution 
of  one  parasite.  These  different  stages  are  normal  and  regular,  and 
not  accidental  or  chance  forms,  and  for  the  sake  of  convenience  we 
may  summarise  them  seriatim  thus : — 

1.  Early  Form  of  Parasite. — Looking  through  the  microscope,  we 
shall  readily  observe  large  numbers  of  blood  corpuscles,  and  in  some 
of  these,  and  possibly  many  of  them,  there  will  be  apparent  certain 
irregularities.     In   the   first  place,  the   protoplasm  of  the  affected 
corpuscles  is  paler  than  that  of  the  healthy  cells.     Next,  within  the 
protoplasm  will  be  seen  the  parasite  (amcebula),  containing  possibly  a 
few  specks  of  black  pigment,  and  of  more  or  less  irregular  outline, 
sometimes  nearly  filling  the  whole  blood  corpuscle.     This   body  is 
motile,  and  moves  about  like  an  amoeba  inside  the  corpuscle,  in  the 
tertian   fever   with  great  rapidity.      As   it  increases   in   size,  the 
corpuscle  becomes  paler.     The  largest  of  these  spherical  forms  are 
outside  the  cells  (extra-corpuscular  spores,  enhaemospores),  and  move 
about  free  in  the  blood  plasm.     But  the  smaller  ones  are  generally 
inside  the  blood  cells  (intra-corpuscular  amoebula).     They  live  at  the 
expense  of  the  haemoglobin  in  the  corpuscle,  and  ultimately  change 
it  into  pigment  (melanin). 

2.  Concentration  of  the  Pigment. — After  the  parasite  has  gained 
its  mature  form  as  regards  size,  an  increase  and  concentration  of  the 
pigment  occurs.     The  body  of  the  parasite  now  fills  the  corpuscle, 
and  the  pigment  which  before  existed  in  specks,  or  diffusely,  becomes 
gathered  together  towards  the  centre  of  the  parasite. 

3.  The  third  change  in  the  evolution  of  the  amosbula  is  segmenta- 
tion.   By  this  process  the  parasite  splits  up  into  segments;    the 
tertian  fever  forming  much  smaller  and  more  numerous  segments 
than  the  quartan.     This  segmentation  gives  rise  to  what  is  known 
as  .the  "  rosette  body." 

4.  In  reality  these  segments  are  sporocytes,  new  amoeboid  bodies, 
which,  by  the  rupture  of  the  ea ten-out  corpuscle,  become  diffused 
freely  in  the  blood.     Many  of  these  "  spores "  are  supposed  to  pass 
to  the  spleen,  some  are  absorbed  by  phagocytes  or  scavenging  cells, 

*  Deutsche  Medicinische  Wochensfihrift >  February  1900. 


FORMS  OF  MALARIAL  FEVER  373 

but,  in  a  few  hours,  many  others  reappear  in  the  blood  and  inaugurate 
another  stage. 

5.  The,  Infection  of  the  Corpuscles. — The  spores  now  attach  them- 
selves to  healthy  blood  corpuscles,  slowly  pass  into  their  interior, 
and  set  up  a  precisely  similar  series  of  changes;  the  actively 
amoeboid  stage,  the  increase  of  size  and  pigmentation,  the  con- 
centration of  the  pigment,  the  mature  form,  and  the  segmenta- 
tion resulting  in  the  rosette  body,  and  eventual  escape  of 
sporocytes.  In  this  way  the  multiplication  of  the  parasite  is 
carried  on  in  the  human  host.  This  is  known  as  the  Cycle  of 
Golgi.  Each  paroxysm  of  malaria  is  related  to  the  evolution  cycle 
of  a  generation  of  these  parasites — probably  many  millions  in 
number — the  commencement  of  each  paroxysm  coinciding  with  the 
maturation  of  a  generation  of  parasites.  The  severity  of  a  paroxysm 
in  a  given  type  of  fever  is  also  in  direct  relation  to  the  number  of 
parasites  in  the  blood.  It  does  not  necessarily  follow  that  the 
gravity  of  the  case  is  in  proportion  to  the _in tensity  of  the  paroxysms. 

Malaria  is  characterised  by  marked  intermittency,  which  is  usually 
divided  clinically  into  three  leading  forms : — 

(a)  Quartan,  4epending  upon  a  parasite  which  takes  seventy-two 
hours  to  pass  through  its  cycle  of  development,  and  produces  fever 
every  third  day.  The  corpuscles  invaded  do  not  become  so  much 
decolorised,  hypertrophied,  or  altered  in  shape  as  in  other  forms. 
The  parasite  shows  distinctly  less  amoeboid  movement,  and  is  not 
so  delicate  in  structure  or  definition  as  in  the  Tertian  varieties, 


FIG.  29.— Quartan  Malaria  Parasite. 

though  it  carries  a  large  amount  of  dark  brown,  pigmented  material, 
which  is  coarse  in  grain.  The  developed  sporocyte  has  what  is 
described  as  a  "  daisy-head  "  appearance.  The  six  to  fourteen  spores 
are  rounded  in  form,  and  possess  a  well-defined  nucleus.  Quartan 
fever  is  relatively  much  more  common  in  temperate  and  subtropical 
latitudes  than  in  the  tropics. 

(b)  Benign  or  Mild  Tertian. — In  this  fever  the  parasite  takes 
forty-eight  hours  to  complete  its  cycle.  The  amoebula  is  actively 
motile  inside  the  corpuscle,  giving  rise  to  great  and  rapidly-changing 


374 


THE  ETIOLOGY  OF  TROPICAL  DISEASES 


irregularities  in  the  condition  of  the  corpuscle,  which  becomes  swollen, 
pale  in  colour,  and  may  show  deeply-stained  "  spots."  The  pigment 
granules  are  finer  than  in  the  quartan  parasite.  The  final 


FIG.  30.— Tertian  Malaria  Parasite. 

decolonisation  of  the  corpuscle  is  very  marked.  The  "rosette 
body"  or  sporocyte  in  this  species  is  composed  of  some  fifteen  to 
twenty-five  spores,  small,  smooth,  and  oval;  the  gametes  are 
spherical.  The  benign  tertian  parasite  is  probably  the  commonest 
form  found  in  malaria,  and  is  widely  distributed  over  the  globe. 

(c)  The  Malignant  Infections  (sestivo  -  autumnal,  malignant 
quotidian,  malignant  tertian).  The  amcebulce  in  these  conditions 
are  much  smaller  than  in  the  benign  types,  but  may  occur  in  pro- 
digious numbers,  and  their  movements  are  very  active.  The  organism 
causes  considerable  modifications  in  the  shape  and  size  of  the  corpuscle, 
which  has  a  tendency  to  shrivel.  It  is  not  filled  by  the  parasite  in  the 
same  degree  as  in  the  other  forms.  Sporulation  occurs  in  the  spleen 


FIG.  31.— Malignant  Malaria  Parasite. 


and  other  internal  organs,  and  not  in  the  blood,  and,  therefore,  the 
sporocytes  in  this  form  are  not  found  in  the  blood  in  the  usual  way. 
The  most  distinctive  feature  of  all  is  that  the  malignant  parasites 
(gametes)  form  "crescents,"  and  attack  a  larger  proportion  of  red 
corpuscles  than  in  the  other  forms.  The  classifications,  minor 


FORMS  OF  MALARIAL  FEVER  375 

subdivisions,  and  clinical  nomenclature  have  passed  through  a  variety 
of  changes.  The  three  old  divisions  have  been  retained  here  for 
convenience.  Sir  Patrick  Manson  has  suggested  the  following 
classification : — 

4    TJ     •  f  Quartan  1         In  which  the  parasites  do 

A.  Benign    .    ^Jertian  )   =          not  form  crescents. 

(  Quotidian — with  pigmentation       ^          In  which  the  para- 

B.  Malignant  -I  Quotidian — without  pigmentation  j-   =         sites    do    form 

( Sub-tertian  )  crescents. 

Now  in  some  forms  of  malarial  fever,  namely,  the  malignant 
infections,  the  spherical  bodies  or  mature  form  immediately  prior  to 
segmentation  into  rosette  bodies,  do  not  actually  show  segmentation, 
but  assume  the  form  of  crescents  lying  inside  the  blood  corpuscle,  the 
haemoglobin  of  which  has  been  absorbed.  Between  the  poles  of  the 
crescent  may  be  seen  the  membrane  of  the  blood  corpuscle,  the 
crescent  being  folded  somewhat  on  itself.  These  crescents  represent 
the  form  of  the  parasite  which  requires  to  enter  the  body  of  the 
mosquito  in  order  to  attain  development.  The  crescents  do  not,  as 
a  rule,  appear  in  the  blood  until  about  one  week  from  the  onset  of 
the  fever,  and  are  the  first  stage  of  the  extra-corporeal  phase  of  the 
parasite.  They  are  termed  the  gametocytes,  and  are  of  two  kinds, 
male  and  female.  It  will  be  necessary  to  follow  the  development  of 
each  kind  separately. 

The  mierog'ametoeyte,  or  male  gamete,  is  the  parasite  in  crescent 
form,  with  the  delicate  membrane  of  the  containing  blood  corpuscle 
at  first  investing  it,  as  described  above.  These  crescents  are  hyaline 
in  appearance,  and  the  motionless  pigment  is  loosely  arranged.  The 
crescent  eventually  absorbs  or  exhausts  the  blood  corpuscle,  and 
becomes  a  free  body  in  the  blood  serum.  Next,  it  changes  by 
becoming  kidney-shaped,  then  round  at  the  poles,  thicker,  more 
ellipsoidal,  and  eventually  spherical.  The  pigment  granules  now 
become  mobile,  and  eventually  assume  most  active  movement  and 
become  diffused  throughout  the  whole  sphere,  and  almost  immedi- 
ately thereafter  the  sphere  itself  becomes  agitated,  and  from*  its 
circumference  shoot  out  long  flagella  (microgametes).  This  flagellated 
parasite  is  a  weird -looking,  "  octopus-like  "  body,  with  long  lashing 
tentacle-like  flagella,  and  containing  in  its  centre  a  mass  of  moving 
pigment  granules.  The  flagella  or  microgametes  are  of  the  nature 
of  spermatozoa,  and  fulfil  a  similar  function,  and  are  in  length  some 
three  or  four  times  the  diameter  of  the  microgametocyte.  They  are 
unpigmented,  and  may  bear  at  their  extremities  bulbous  swellings. 
They  break  off  and  become  free  in  the  blood,  continuing  their 
active  movements. 

The  flagellated  body  may  be  produced  from  free  spheres  of  the 


376  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

quartan  and  benign  tertian  parasite  (which  do  not  produce  crescents) 
as  well  as  from  crescents.  But  it  is  never  seen  in  fresh  blood 
immediately  after  being  drawn  from  the  body.  It  only  appears  after 
the  blood  has  left  the  body  for  twenty  minutes  or  half  an  hour.  Such 
a  striking  transformation  of  a  free  sphere  or  a  crescent  is  evidently 
a  stage  of  great  importance,  and  two  different  explanatory  theories 
have  been  advanced  to  account  for  it.  Some  have  held  it  to  be  a 
degenerative  change  in  the  parasite — that  the  coldness  of  the 
outside  air  has  killed  it,  that  its  contortions  and  wriggling  flagella 
are  but  its  death  struggles,  and  that  the  active  movement  of  its 
pigment  particles  are  but  Brownian  movements  of  dead  granules. 
Other  authorities,  and  particularly  Sir  P.  Manson,  have  declared  the 
flagellated  body  to  be  a  vital  evolutionary  change — a  normal  step  in 
the  life  of  the  parasite,  the  first  stage  in  its  life-history  outside  the 
human  body,  the  extra-corporeal  homologue  of  the  intra-corporeal 
sporulating  body.  It  is  now  agreed  that  these  microgametes  or 
flagella  are  the  essential  sporulating  bodies  of  an  extra-corporeal 
phase,  and  that  their  function  is  the  impregnation  of  the  female 
gametocyte. 

The  maerog'ametoeyte,  or  female  gamete,  is  the  second  kind  of 
gamete,  and  starts  its  course  in  much  the  same  way  as  the  male  cell. 
It  also  is  a  crescent  inside  the  blood  cell,  which  it  eventually 
breaks  down,  and  thus  becomes  free  in  the  blood  serum. 
Instead  of  being  hyaline  it  is  granular,  and  the  pigment  is 
situated  more  centrally.  It  eventually  becomes  ellipsoidal,  and 
then  spherical.  The  protoplasm  of  the  female  crescents  stains 
more  deeply  than  that  of  the  male  crescents;  the  pigment  is 
more  closely  grouped  together,  generally  in  ring  form,  in  the 
centre  of  which  will  be  seen  one  or  occasionally  two  large 
masses  of  chromatin.  At  its  maturity  as  a  macrogametocyte,  two 
small  polar  bodies  or  excrescences  or  papilla  are  seen  on  its  circum- 
ference, and  it  is  at  this  site  that  impregnation  by  the  free  flagellum 
(male  cell  or  microgamete)  is  effected.  The  .result  is  the  zygote,  or 
travelling  vermicule.  In  1897,  MacCallum  observed  this  impregna- 
tion actually  taking  place  in  a  case  of  human  malaria,  and  others 
have  observed  it  in  one  of  the  malaria-like  organisms  of  birds,  the 
halteridium.  After  its  entry  into  the  female  cell  the  flagellum 
became  quiescent,  and  the  pigment  became  collected  at  the  posterior 
end  of  the  cell,  which  then  assumed  the  shape  of  a  spear  head,  and 
became  the  actively-motile  zygote. 

The  Mosquito  Phase. — Just  as  the  segmentation  body  eventually 
splits  up  into  spores  for  the  further  propagation  of  the  parasite  in 
the  blood  of  the  malarial  patient,  so  the  flagellated  body  provides 
for  the  propagation  of  the  parasite  in  some  living  host  outside  the 
human  body ;  for,  as  is  well  known,  parasites  pass  from  one  host  to 


THE  MOSQUITO  THEORY 


37 


another.  There  is  now  complete  evidence  to  show  that  the  host  is 
the  Mosquito.  It  is  therefore  necessary  to  consider  briefly  the 
outstanding  features  of  the  Mosquito  Phase. 

The    Mosquito    is    widely    distributed,    especially    in    tropical 


FJG.  32.— Anopheles  maculipennis  9  (Meigen). 

countries,  and  is  generally  classified  under  the  genus  Culicidoe,  of 
which  probably  a  large  number  of  varieties  exist.  The  Culex  is  the 
commonest  species.  Its  larvae  live  almost  everywhere  in  warm 
countries,  inhabiting  any  pot,  tub,  well,  cistern,  broken  bottle,  or, 


378 


THE  ETIOLOGY  OF  TROPICAL  DISEASES 


indeed,  anything  in  which  a  little  water  can  lodge.  They  are 
almost  domestic  animals.  The  palpi  are  short,  the  wings  unspotted, 
the  proboscis  thin,  the  thorax  large,  and  the  larvae  have  breathing 
tubes.  They  prefer  to  lie  in  artificial  collections  of  water.  When  at 
rest,  for  example  on  a  wall,  the  body  of  Culex  is  found  parallel  to  the 
wall  (see  fig.  33).  The  rarer  species,  and  that  which  has  been  proved 
to  be  the  host  of  the  malaria  parasite,  is  the  Anopheles.  This  differs  in 
various  essential  particulars  from  the  Culex.  In  Anopheles  the  body 
is  slim,  but  the  proboscis  long  and  thick,  the  palpi  are  long,  and  the 


FIG.  33.— Diagram  of  Culex  and  Anopheles  on  Wall  (Ross). 

wing  is  "dappled"  with  dark  spots  on  its  anterior  margin.  The 
larvae  have  no  breathing  tubes,  and  lie  horizontally  (not  vertically 
as  in  Culex)  in  the  water  of  puddles,  looking  like  bits  of  brown 
stick  or  thorns  floating  on  the  surface.  When  at  rest  on  a  wall,  the 
axis  of  its  body  is  almost  at  right  angles  to  the  wall,  so  that  the 
head  of  the  Anopheles  is  directed  towards  the  wall,  whilst  the  body 
projects  out  into  the  room.  Culex  has  been  briefly  designated  a 
"  pot-breeding "  mosquito,  whilst  one  of  the  features  of  Anopheles  is 
that  it  is  "  puddle-breeding."  Its  favourite  haunts  are  slow,  small 
streamlets  containing  algae ;  small,  shallow,  natural  puddles  with 
confervoid  growths  in  the  water ;  or  stagnant  and  fairly  permanent 


THE  MOSQUITO  THEORY  379 

collections  of  rain-water.  The  larvse  cannot  live  when  the  puddles 
in  which  they  breed  dry  up.  Nor  does  Anopheles  appear  to  favour 
swamps  containing  deep  water.  The  puddles  they  select  are  in 
immediate  proximity  to  houses,  where  the  adult  mosquitoes  may 
frequently  pass  to  find  human  beings  or  cattle,  from  whom  they 
may  derive  their  nourishment.  As  is  well  known,  it  is  the  female 
mosquito  which  is  the  blood-sucker.  After  she  has  filled  herself 
with  blood,  she  retires  to  some  dark,  sheltered  spot  near  a  stagnant 
puddle,  and  after  a  few  days  deposits  her  eggs  (from  200-400  in 
number)  in  a  mass  on  the  surface  of  the  water.  Then  she  dies  and 
falls  into  the  water  beside  her  eggs.  The  eggs  give  rise  (sometimes 
in  sixteen  hours  time)  to  the  tiny  swimming  larvse,  which  feed 
greedily  and  grow  rapidly,  shed  their  skins,  and  become  nymphce  or 
pupce.  Eventually,  the  shell  of  the  nympha  cracks  along  its  dorsal 
surface,  and  the  young  mosquito  emerges.  Standing  on  the  raft  of 
its  empty  pelt,  it  dries  its  wings  and  flies  away.*  Very  soon  it  also 
lays  eggs.  The  entire  cycle  from  egg  to  egg  is  about  fifty  days. 
The  three  conditions  necessary  for  the  multiplication  of  malarious 
mosquitoes  are  (a)  high  atmospheric  temperature,  75°  to  104°  F. ; 
(&)  collections  of  water,  fresh  or  brackish ;  and  (c)  the  presence  in 
the  breeding  pools  of  low  forms  of  animal  and  vegetable  life. 

Having  now  gathered  the  outstanding  facts  concerning  the 
mosquito,  we  may  return  to  the  part  which  it  plays  in  the  propaga- 
tion of  malaria.  The  method  which  nature  elects  for  the  liberation 
of  any  given  parasite  is  generally  one  that  is  reasonably  regular  and 
frequent  in  its  operations.  Hence,  it  occurred  to  Laveran  and 
Manson  that  as  the  malarial  organism  is  a  passive  blood  parasite, 
its  escape  from  the  human  body  might  be  effected  on  the  same 
principle  that  the  escape  of  the  passive  muscle  parasites  is  effected. 
As  the  latter  obtain  their  opportunity  by  being  swallowed  by  some 
flesh  eater,  Dr  Manson  reasoned  that  the  blood  parasite  might  obtain 
similar  liberty  by  being  swallowed  by  some  blood-eater,  some 
suctorial  insect  such  as  the  sand-fly  or  mosquito.  He  was  still 
further  led  to  the  mosquito  theory  owing  to  the  parallel  conditions 
which  he  had  already  found  to  exist  in  the  case  of  the  analogous 
mosquito  phase  of  Filaria  sanguinis.^  The  mosquito  phase  then  is 
the  extra-corporeal  stage  of  the  life-history  of  the  malaria  parasite 
which  takes  place  within  the  body  of  the  mosquito,  and  we  may 
now  briefly  follow  the  course  of  events  in  the  further  development 
of  the  flagellated  body  inside  the  mosquito. 

Whilst  the  mosquito  theory  was  largely  the  suggestion  of   Sir 

*  For  further  particulars  as  to  mosquito  Anopheles,  see  Brit.  Med.  Jour.  1901, 
i.,  p.  195;  Jour,  of  Hygiene,  1901,  i.,  pp.  4-77,  269,  and  451,  also  vols.  1902  and 
1903. 

t  Goulstonian  Lectures,  1896.  Sir  Patrick  Manson.  Brit.  Med.  Jour.,  1896,  i., 
641  el  seq.,  and  1900,  i.,  328. 


380 


THE  ETIOLOGY  OF  TROPICAL  DISEASES 


P.  Manson,  the  practical  investigation  and  final  elucidation  of  it 
were  in  great  measure  due  to  the  patient  observance  and  skill  of 
Eonald  Koss,  at  that  time  a  Surgeon-Major  in  the  Indian  Medical 


FIG.  34. — SCHEME  SHOWING  HUMAN  AND  MOSQUITO  CYCLES  OF  THE  MALARIA 
PARASITE  (MANSON). 

1-8,  the  human  or  endogenous  or  asexual  cycle ;  9-24,  the  mosquito  or  exogenous  or  sexual  cycle.  1, 
Normal  red  blood  corpuscle ;  2-5,  blood  corpuscles  containing  an  amcebula ;  6-8,  sporocytes  ; 
9,  young  gametocyte ;  11,  13,  15,  17,  microgametocytes  or  male  gametes  ;  10,  12,  14,  16,  macro- 
gametocytes  or  female  gametes;  18,  female  gametocyte  being  impregnated  by  microgametes ; 
19,  travelling  vermicule;  20,  young  zygote ;  21,  22,  zygotomeres ;  23,  blastophore ;  24,  mature 
zygote. 

Service.  His  chief  contribution  to  the  solution  of  the  problem  was 
the  discovery  of  the  malarial  parasite  in  the  tissues  of  the  mosquito. 
He  found  that  in  the  body  of  mosquitoes  fed  upon  malarial  human 
blood  70  per  cent,  of  the  crescent  forms  of  the  parasite,  as  we  have 
seen  one  of  the  latent  forms  of  the  flagellated  body,  were  transformed 


THE  MOSQUITO  THEORY  381 

into  the  flagellated  body.  This  transformation  occurred  in  a  pro- 
portion of  instances  very  much  greater  than  occurs  in  malarial 
blood  spread  in  the  ordinary  way  on  a  slide  and  exposed  to  the  air. 
He  found,  also,  that  the  flagella  broke  away  from  the  flagellated 
body,  yet  he  was  unable  to  trace  what  became  of  the  free  flagella.  But 
in  a  "dapple-winged"  mosquito  (anopheles)  fed  in  the  same  way 
with  malarial  blood  containing  crescents  (summer-autumn  fever), 
he  discovered,  embedded  in  the  tissues  of  the  stomach  wall,  certain 
peculiar  oval  cells  containing  the  same  pigment  as  the  malarial 
parasite  in  the  human  blood.  This  was,  in  fact,  the  extra-corporeal 
form  of  the  human  parasite  after  impregnation,  namely,  the  zygote. 
Next  he  discovered  pigmented  cells  in  the  body  tissues  of  mosquitoes 
fed  upon  sparrows'  blood,  affected  with  a  similar  parasitical  condition 
to  malaria  (proteosoma) ;  and  from  one  step  to  another  he  demon- 
strated the  evolution  of  the  mosquito  phase  of  these  parasites. 
Eoss  made  it  evident  that  the  cycle  of  extra-corporeal  development 
of  the  parasite,  as  we  have  already  seen,  is  carried  on  inside  the 
body  of  the  mosquito.  After  malarial  blood  is  shed  or  swallowed 
by  the  mosquito,  the  changes  already  described  take  place.  Practi- 
cally, all  the  crescents  become  spheres  within  a  few  minutes  of  being 
taken  into  the  stomach  of  the  mosquito,  then  the  male  gametes 
become  flagellated,  and  the  female  gametes  become  impregnated. 
According  to  Eoss,  the  condition  which  brings  about  the  transfor- 
mation of  crescents  into  flagellated  bodies  is  not  low  temperature, 
nor  exposure  to  air,  nor  contact  with  the  wall  of  the  mosquito's 
stomach,  but  abstraction  of  the  water  from  the  serum  of  the  blood. 

However  that  may  be,  the  changes  resulting  from  impregnation 
result  in  the  mosquito's  stomach  in  the  production  of  the  zygote  or 
fertilised  cell.  This  body  is  a  travelling  vermicule,  and  on  or  about 
the  second  day  it  penetrates  the  stomach  wall  and  becomes  encysted 
between  the  muscle  fibres.  The  number  of  zygotes  produced  in  the 
mosquito  after  its  feed  on  malarial  blood  varies  widely,  sometimes 
being  few,  sometimes  many.  In  the  enlarging  encysted  cell  there 
now  come  to  be  developed  a  number  of  cells  known  as  zygotomeres, 
which  as  development  proceeds  become  blastophores  filled  with 
filiform  spore  cells  (sporozoits  or  germinal  rods  or  zygotoblasts). 
Ultimately,  the  zygote  becomes  thus  transformed  into  a  cyst  (sporo- 
cyst)  packed  full  of  zygotoblasts.  When  fully  developed,  at  about 
the  eighth  or  ninth  day  after  the  mosquito  ingested  the  malarial  blood, 
the  sporocyst  measures  about  60  micromillimetres  in  diameter. 
About  the  twelfth  day  it  bursts,  discharging  the  zygotoblasts,  which 
are,  of  course,  "  spores  "  or  reproductive  elements,  into  the  body  cavity 
and  fluid  of  the  mosquito,  and  spreading  from  thence  they  become 
accumulated  in  the  large  veneno-salivary  gland,  and  are  thus  in  a 
position  to  be  injected  along  with  its  secretion  into  the  human 


382  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

subject  next  bitten.  These  zygotoblasts  are  the  actual  source  then 
of  infection  of  man,  and  on  arriving  in  the  human  blood  the  parasite 
(as  spores)  attacks  the  blood  cells,  and  thus  commences  the  intra- 
corporeal  or  human  phase  described  above.  The  mosquito  phase 
occupies  a  time  varying  between  six  to  sixteen  days  or  longer, 
depending  on  temperature  and  other  factors. 

Such,  in  outline,  is  the  mosquito  theory  of  malaria.  No  one 
supposes  that  the  last  word  has  been  said.  But  sufficient  is  now 
known  to  make  it  certain  that  the  mosquito  phase  is  a  fact  of 
essential  importance  in  the  conveyance  of  the  disease  to  man.  In 
the  first  place,  the  malarial  parasite  has  been  found  repeatedly  in 
the  body  of  the  mosquito,  and  in  the  second  place  the  crucial 
experiment  of  inoculation  has  been  performed,  and  has  yielded  a 
positive  result.  Infected  mosquitoes  were  brought  from  the  Roman 
Gampagna,  and  Dr  Thorburn  Mason  and  Mr  George  Warren  con- 
sented to  be  bitten,  and  thus  contracted  malaria.  There  yet  remain 
gaps  to  be  filled  up  in  our  knowledge  of  the  disease,  but  there  can 
be  little  doubt  that  future  work  will  further  establish  and  elaborate 
the  principles  of  the  mosquito  theory,  and  the  lines  of  prevention 
will  of  necessity  follow  the  new  facts  now  proved.* 

As  concerns  preventive  medicine,  the  new  facts  may  be  sum- 
marised in  three  propositions : — (1)  Malaria  is  caused  by  a  number 
of  microscopical  parasites  which  live  and  propagate  themselves  in 
the  blood.  (2)  These  parasites  are  carried  from  infected  persons  to 
healthy  ones  by  the  agency  of  the  genus  of  mosquitoes  termed 
Anopheles.  (3)  These  mosquitoes  breed  chiefly  in  shallow  and 
stagnant  terrestrial  waters. 

Examination  of  Malarial  Blood  (see  Appendix,  p.  485). 

The  Prevention  of  Malaria 

The  new  knowledge  respecting  malaria  indicates  the  only 
adequate  preventive  methods.  The  malarial  parasite  gains  access 
to  the  human  subject  by  means  of  mosquito  bites,  and,  as  far  as 
is  known,  in  no  other  way.  Hence  the  methods  of  prevention  must 
be  directed  mainly  against  the  mosquito  : — 

1.  The  prevention  of  mosquito-breeding. 

2.  The  destruction  of  mosquitoes. 

3.  Avoidance  of  being  bitten  by  mosquitoes. 

4.  The  use  of  quinine. 

1.  The  Prevention  of  Mosquito-breeding-.—In  order  to  prevent 

the  breeding  of  mosquitoes,  it  is  necessary  to  eradicate  all  possible 

breeding-places.     As  we  have  seen,  such  places  are  tanks,  cisterns, 

vessels   of  stagnant   water,  ditches,  small  pools,  buckets,  cocoa-nut 

*  For  full  account  of  malaria,  see  Tropical  Diseases  (Manson),  1903,  pp.  1-173. 


PREVENTION  OF  MALARIA  383 

shells,  tins,  cans,  pots,  etc.,  wherever  stagnant  water  readily  collects, 
especially  near  houses.  The  larvae  of  Culex  float  when  at  rest  on  the 
surface  of  the  water,  suspended  by  their  tails  and  with  their  heads 
hanging  downward;  when  disturbed,  they  wriggle  to  the  bottom. 
The  larvae  of  Anopheles  float  flat  on  the  surface  like  small  sticks,  and 
when  disturbed  they  wriggle  on  the  surface  with  a  backward  skating 
movement.  The  former  are  usually  present  in  artificial  collections 
of  water,  such  as  pots,  broken  bottles,  cans,  etc.,  whilst  Anopheles 
prefer  natural  collections  of  water,  "chiefly  rain-water  puddles  which 
do  not  dry  up  quickly,  or  which  contain  green  water-weed.  Such 
being  the  breeding-places,  prevention  is  simple.  Collections  of 
stagnant  water  must  not  be  permitted.  Search  must  be  made  for 
them,  vessels  must  be  emptied  and  puddles  brushed  out  with  a 
broom,  and  small  pools  drained  and  filled  in.  Water  must  not  be 
allowed  to  collect  anywhere  near  the  house.  Land  drainage  is  an 
obvious  preventive  of  the  first  importance. 

Cisterns  and  similar  necessary  collections  of  water  may  be 
protected  by  paraffin  or  petroleum,  for  these  by  lying  on  the 
surface  of  the  water  prevent  the  pupae  of  the  mosquito  from  reaching 
the  surface  at  the  time  of  transformation.  When  water  is  required, 
it  may  be  drawn  off  from  the  bottom  of  the  cistern.  The  surface  of 
paraffin  may  be  renewed  once  a  fortnight  or  oftener  if  necessary. 
Paraffin  (kerosene  oil)  also  acts  as  a  culicicide,  destroying  the  larvae 
by  choking  their  air  tubes.  The  essential  condition  in  any  scheme 
for  the  sanitary  improvement  of  a  malarious  region  is  that  the  eggs, 
larvae,  and  nymphae  of  the  mosquito  should  be  exterminated  in  that 
region.  Covering  small  collections  of  water  with  healthy  soil,  accom- 
panied by  thorough  drainage,  inasmuch  as  they  remove  at  the  same 
time  both  the  water  and  the  atmospheric  air,  the  two  indispensable 
elements  of  mosquito  life,  are  the  best  preventive  methods.* 

2.  The  Destruction  of  Mosquitoes.— Smoke  from  a  wood  fire  or 
damp  tobacco  leaves,  or  sulphur,  or  other  gaseous  disinfectants  may 
be  used  for  this  purpose.     Kerosene  may  be  used  as  recommended 
above  for  killing  the  larvae.     Individual  mosquitoes  should  be  killed 
whenever  possible. 

3.  Avoidance  of  being1  Bitten  by  Mosquitoes. — For  the  protec- 
tion of  the  person  from  attack  by  mosquitoes,  there  are  a  variety  of 
contrivances,  from  mosquito  nets  to  mosquito-proof  houses.    Mosquito 
nettings  on  the  bed  should  invariably  be  used  in  malarious  countries. 
There  are  several  points  as  to  the  effectual  use  of  such  nets.     In  the 
first  place,  the  net  should  be  square,  should  be  hung  inside  a  frame- 
work, tucked  carefully  under  the  mattress  all  round  and  not  allowed 
to  hang  down,  and  stretched  tight  so  as  to  allow  air  to  pass  in  easily. 

*  See  also  Brit.  Med.  Jour.  1900,  vol.  i.,  pp.  300-306  (Celli) ;  and  1901,  vol.  i., 
pp.  193-203  and  p.  240. 


384  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

In  the  second  place,  the  roof  should  be  made  of  netting  similar  to  the 
other  parts  of  the  net,  and  not  of  cloth.  Thirdly,  during  the  day, 
when  the  net  is  not  being  used,  it  should  be  hung  up  in  such  a  way 
as  to  prevent  mosquitoes  entering  it.  Fourthly,  the  mesh  of  the  net 
should  be  sufficiently  fine  to  effect  its  purpose,  and  should  contain 
no  rents,  holes,  or  other  apertures.  Where  punkahs  are  available, 
they  should  swing  above  the  mosquito  net. 

Again,  habits  of  life,  especially  temperateness  and  moderation, 
not  sleeping  in  the  open  air,  living  as  far  as  possible  in  healthy 
houses,  not  frequenting  native  quarters  after  sunset,  and  not 
associating  with  native  children,  are  methods  by  which  to  avoid  being 
bitten.  It  is  now  well  known  that  in  malarious  towns  and  districts 
the  great  majority  of  native  children  harbour  the  malarious  parasites 
in  their  blood,  and  therefore  segregation  is  a  necessary  preventive 
method.  Europeans'  houses  should  be  built  at  a  distance  from  the 
native  quarters. 

The  experiments  of  Sambon  and  Low,  of  Celli,  of  G-rassi,  of  Fermi, 
and  Tonsini,  and  of  the  Red  Cross  Society  of  Italy,  have  demonstrated 
beyond  all  question  that  it  is  practicable  to  construct  habitable  houses 
which  shall  be  mosquito-proof.* 

Lastly,  probably  some  protection  is  obtained  by  means  of 
perfumes,  washes,  pomades,  soaps,  etc.,  though  these  should  not  be 
relied  upon.  Certainly  flannel  clothing  is  a  great  advantage. 

4.  Quinine. — When  it  is  too  late  for  preventive  measures  the 
time  has  come  for  isolation,  disinfection  of  rooms  containing  infected 
mosquitoes,  and  treatment.  A  person  with  malaria  is  always  a  risk 
to  other  persons,  and  should  be  isolated  as  far  as  practicable.  Infected 
rooms  should  be  disinfected  with  gaseous  disinfectants,  such  as 
sulphur  or  formic  aldehyde.  Quinine  is  the  specific  remedy  in 
treatment.  It  acts  not  only  as  an  antipyretic  but  as  a  specific  drug, 
destroying  the  parasite  in  the  blood.  For  an  ordinary  intermittent 
fever  the  dose  of  quinine  may  be  10  grains,  given  when  the  sweating 
stage  commences,  followed  by  5  grains  every  six  or  eight  hours  for  a 
week,  and  with  a  view  of  preventing  relapse  5  grains  three  times 
every  fifth,  sixth,  or  seventh  day  for  two  or  three  months.  Five  to 
ten  grains  of  quinine  twice  a  week  or  oftener,  tends  to  prevent  or 
abate  incipient  malarial  infection. 

2.  Cholera 

This  word  is  used  to  denote  a  group  of  diseases  rather  than  one 
specific  well-restricted  disease.  In  recent  years  it  has  become 
customary  to  speak  of  Asiatic  cholera  and  British  cholera,  as  if, 
indeed,  they  were  two  quite  different  diseases.  But,  as  a  matter 

*  Practitioner,  March  1901,  p.  262. 


CHOLERA  385 

of  fact,  we  know  too  little  as  yet  concerning  either  form  to  dogmatise 
on  the  matter.  Until  1884  practically  nothing  was  known  about 
the  etiology  of  cholera.  In  that  year,  however,  Koch  greatly  added 
to  our  knowledge  hy  isolating  a  spirillum  from  the  intestine,  and  in 
the  dejecta  of  persons  suffering  from  the  disease. 

Cholera  has  its  home  in  the  delta  of  the  Ganges.  From  this 
endemic  area  it  spreads  in  epidemics  to  various  parts  of  the  world, 
often  following  lines  of  communication.  Cholera  is  generally 
conveyed  by  means  of  water.  It  is  a  disease  which  is  characterised 
by  acute  intestinal  irritation,  manifesting  itself  by  profuse  diarrhoea 
and  general  systemic  disturbance  accompanied  by  collapse,  cramps, 
cardiac  depression,  subnormal  temperature,  and  suppression  of  urine. 
The  incubation  period  varies  from  only  a  few  hours  to  several  days. 
In  the  intestine,  and  setting  up  its  pathological  condition,  are  the 
specific  bacteria,  in  the  general  circula- 
tion their  toxic  products  bringing  about 
the  systemic  changes. 

The  spirillum  of  Asiatic  cholera 
(Koch,  1884)  generally  appears  in  the 
body  and  in  artificial  culture,  broken 
into  bacillary  elements  known  as 
"  commas."  These  are  curved  rods 
with  round  ends,  showing  an  almost 
equal  diameter  throughout,  and  some- 
times united  in  pairs  or  even  in  chains 
(spirillum).  The  latter  rarely  occurs  in 
the  intestine,  but  may  be  seen  in  fluid 

T,  rrn  •  i       £        ir      ~u>  FIG.  35. — Diagram  of  the  Comma 

cultures.     The  common  site  for  Koch  s  Bacillus  of  cholera. 

comma  is  in  the  intestinal  wall,  crowd- 
ing the  tubules  of  the  intestinal  glands  situated  between  the  epi- 
thelium and  the  basement  membrane,  abundant  in  the  detached 
flakes  of  mucous  membrane,  and  free  in  the  contents  of  the  intes- 
tine. The  bacilli  are  present  in  enormous  numbers,  and  lie  usually 
with  their  long  axes  in  the  same  direction,  giving  the  "fish  in 
stream "  appearance  (Koch).  The  bacilli  do  not  occur  in  the  blood, 
nor  are  they  distributed  in  the  organs  of  the  body.  They  occur 
mostly  in  the  lower  intestine. 

The  bacillus  is  actively  motile,  and  possesses  at  least  one  terminal 
flagellum.  The  organism  is  aerobic,  and  liquefies  gelatine.  It 
stains  readily  with  the  ordinary  aniline  dyes,  but  does  not  stain  by 
Gram's  method.  It  does  not  produce  spores,  though  certain  refractile 
bodies  inside  the  protoplasm  of  the  bacillus  in  old  cultures  have 
been  regarded  as  such.  The  virulence  of  the  bacillus  is  readily 
attenuated,  and  both  the  virulence  and  morphology  appear  to  show 
in  different  localities  and  under  different  conditions  of  artificial 

2  B 


386  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

cultivation  a  large  variety  of  involution  forms.  Unless  the  organism 
is  constantly  being  sub-cultured,  it  readily  dies.  Acid,  even  the  '2 
per  cent,  present  in  the  gastric  juice,  readily  kills  it.  Prolonged 
drying,  or  heating  to  55°  C.  for  sixty  minutes,  or  treatment  with 
weak  chemicals  has  the  same  effect.  The  bacilli,  however,  have 
comparatively  high  powers  of  resistance  to  cold.  Unless  examined 
by  the  microscope  in  a  fresh  and  young  stage,  it  is  difficult  to 
differentiate  Koch's  comma  from  many  other  curved  bacilli. 

Its  characters  in  culture  are  not  always  distinctive.  Microscopic- 
ally, the  young  colonies  in  gelatine  appear  as  cream-coloured,  irregu- 
larly round,  and  granular.  Liquefaction  sets  in  on  the  second  day, 
producing  a  somewhat  marked  "  pitting  "  of  the  medium,  which  soon 
becomes  reduced  to  fluid.  In  the  depth  of  gelatine,  the  growth 
is  very  characteristic.  An  abundant,  white,  thick  growth  exactly 
follows  the  track  of  the  needle,  here  and  there  often  showing  a  break 
in  continuity.  Liquefaction  sets  in  on  the  second  day,  and  produces 
a  distinctive  "  bubble  "  at  the  surface.  The  process  proceeds  steadily, 
at  first  a  funnel-shaped  liquefaction  resulting,  and  then  in  the  course 
of  a  week  or  two  all  the  gelatine  may  be  reduced  to  fluid.  On  agar 
Koch's  comma  bacillus  produces  within  twenty-four  hours  a  thick 
greyish  irregular  growth.  On  potato,  especially  if  slightly  alkaline 
and  incubated  at  37°  C.,  an  abundant  brownish  layer  is  formed. 
Broth  and  peptone  water  are  favourable  media,  and  at  37°  C.  a  general 
turbidity  occurs  with  the  formation  on  the  surface  of  a  pellicle, 
containing  spirilla  in  active  motility.  In  milk  it  rapidly  multiplies, 
curdling  the  medium,  with  production  of  acid.  Unlike  B.  coli,  it 
does  not  form  gas,  but,  like  B.  coli,  it  produces  large  quantities  of 
indol,  and  a  reduction  of  nitrates  to  nitrites.  Hence  the  indol  test 
may  be  applied  by  simply  adding  to  a  peptone  culture  several  drops 
of  strong  sulphuric  acid,  when  in  the  course  of  several  hours,  if  not 
at  once,  there  will  be  produced  a  pink  colour,  "the  cholera  red 
reaction,"  due  to  the  formation  of  nitroso-indol.  Although  the 
bacillus  readily  loses  virulence,  and  its  resistance  is  little,  it 
retains  its  vitality  for  considerable  periods  in  moist  cultures, 
upon  moist  linen,  or  in  moist  soil.  In  cholera  stools  kept  at 
ordinary  room  temperature  the  cholera  bacillus  will  soon  be  out- 
grown by  the  putrefactive  bacteria.  The  same  is  true  of  sewage 
water. 

The  lower  animals  do  not  suffer  from  any  disease  exactly  similar 
to  Asiatic  cholera,  and  hence  it  is  impossible  to  fulfil  the  postulate 
of  Koch  dealing  with  animal  inoculation.  In  this  respect  it  is  like 
the  typhoid  bacillus.  It  is,  however>  provisionally  accepted  that 
Koch's  bacillus  is  the  cause  of  the  disease.  The  four  or  five  other 
bacteria  which  have  from  time  to  time  been  put  forward  as  the  cause 
of  cholera  have  comparatively  little  evidence  in  their  support.  It 


DIAGNOSIS  OF  CHOLERA  387 

is  loss  from  these  and  more  from  several  spirilla  occurring  in  natural 
waters  that  difficulties  of  diagnosis  arise. 

The  reasons  for  believing  Koch's  bacillus  to  be  the  cause  of 
cholera  are  four:  (a)  its  constant  presence  in  cases  of  the 
disease ;  (&)  the  results  of  accidental  infection  with  this  bacillus ; 
(c)  the  agglutinative  and  protective  properties  of  the  serum  of  cholera 
patients ;  and  (d)  the  result  of  Haffkine's  preventive  inoculation. 

There  appears  to  be  evidence  to  show  that  comma  bacilli  may  be 
introduced  into  the  alimentary  canal  without  producing  the  disease, 
unless  there  be  some  injury  or  disease  of  the  wall  of  the  intestine 
(Peiffer).  Desquamation  of  the  intestinal  epithelium  seems  an  essential 
factor  in  the  production  of  the  disease  in  man.  It  need  hardly  be 
added  that  the  bacillus  acts,  like  other  pathogenic  bacteria,  by  the 
production  of  toxins  (Peiffer),  which  appear  to  be  intracellular.  At 
present  very  little  is  known  of  their  chemical  nature.  Brieger 
separated  cadaverin  and  putrescin  and  other  bodies  from  cholera 
cultures,  and  other  workers  have  separated  a  toxalbumin. 

Methods  of  Diagnosis  of  Cholera : — 

1.  The  nature  of  the  evacuations  and  the  appearance   of   the 
mucous    membrane    of    the    intestine    afford   striking   evidence   in 
favour  of  a  positive  diagnosis.     Nevertheless,  it  is  upon  a  minute 
examination  of   the  flakes  and  pieces  of  detached  epithelium  that 
reliance  must  be  placed.     In  these  flakes  will  be  found  abundance 
of  bacilli   having   the   size,  shape,  and  distribution,  of   the   specific 
comma  of  cholera.    The  size  and  shape  have  been  already  referred  to. 
The    distribution    of    comma    bacilli   ("fish    in    stream")   in    the 
flakes  of  watery  stools  is,  when  present,  somewhat  characteristic  of 
Asiatic   cholera,  and  may  greatly  aid  in  a  correct  diagnosis.     But 
unfortunately,  it  is  not  always  present,  and  then  search  for  other 
characters  must  be  made. 

2.  The  appearance  of  cultivation  on  gelatine,  to  which  reference 
has  been  made,  is  of  diagnostic  value,  and  the  growth  on  agar  and  in 
peptone  solution. 

3.  The  "  cholera  red  reaction."     It  is  necessary  that  the  culture 
and  the  sulphuric  acid  be  pure  for  successful  reaction. 

4.  The   intra-peritoneal   injection  in  guinea-pigs  is  followed  by 
abdominal  distention,  subnormal  temperature,  and  other  characteristic 
symptoms. 

5.  Isolation  from  water  is,  according  to  Dr  Klein,  best  accom- 
plished as  follows :  A  large  volume  of  water  (100-500  c.c.)  is  placed 
in  a  sterile  flask,  and  to  it  is  added  so  much  of  a  sterile  stock  fluid 
containing  10  per  cent,  peptone,  5  per  cent,  sodium  chloride,  as  will 
make  the  total  water  in  the  flask  contain  1  per  cent,  peptone  and 
'5  per  cent.  salt.     Then  the  flask  is  incubated  at  37°  C.     If  cholera 
vibrios   are   present  in   the  water,  however  few,  it   will  be   found 


388  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

after  twenty-four  hours'  incubation  that  the  top  layer  contains 
actively  motile  vibrios,  which  can  now  be  isolated  readily  by  gelatine- 
plate  culture. 

6.  To  demonstrate  in  a  rapid  manner  the  presence  of  cholera 
bacilli  in  evacuations,  even  when  present  in  small  numbers,  a  small 
quantity  must  be  taken  up  by  means  of  a  platinum  wire  and  placed 
in  solution  containing  1  per  cent,  of  pure  peptone  and  *5  per  cent, 
sodium  chloride  (Dunham's  solution).     This  is  incubated  as  in  the 
case  of  the  water,  and  in  twelve  hours  is  filled  with  a  turbid  growth, 
which   when    examined   by   means    of    the    hanging    drop    shows 
characteristic  bacilli. 

7.  Pfeiffer's  Test. — Take  a  loopful  of  six  hours'  agar  culture  of 
suspected  cholera  bacilli,  and  add  it  to  1  c.c.  of  ordinary  broth  con- 
taining *001  c.c.  of  anti-cholera  serum  (see  p.  423).     The  mixture 
is  injected  intra-peritoneally  into   a   guinea-pig  of   250   grammes. 
In    20-30    minutes   a   drop   of   peritoneal  fluid  is  withdrawn  and 
examined  microscopically  for  comma  bacilli,  when,  if  the  reaction  is 
positive,  it  will  be  found  that  the  spirilla  have  broken  down  into 
granules. 

3.  Plague 

This  disease,  like  anthrax  and  leprosy,  has  a  long  historical 
record  behind  it.  As  the  Black  Death,  it  decimated  the  population 
of  England  in  the  fourteenth  century,  and  visited  the  country 
again  in  epidemic  form  in  the  middle  of  the  seventeenth  century, 
when  it  was  called  the  Great  Plague.  It  is  highly  probable  that 
these  two  scourges  and  the  recent  epidemic  in  the  East  are  all 
forms  of  one  and  the  same  disease.  As  a  matter  of  fact,  it  is 
difficult  to  be  sure  what  was  the  exact  pathology  of  a  number  of  the 
grievous  ailments  which  troubled  our  country  in  the  Middle  Ages, 
but  from  all  accounts  bubonic  plague  and  true  leprosy  were  amongst 
them.  The  former  came  and  went  spasmodically,  as  is  its  habit; 
the  latter  dragged  through  the  length  of  several  centuries. 

There  are  four  chief  varieties  of  plague :  first,  the  bubonic  form, 
the  most  common  and  typical ;  the  lymph  glands  are  chiefly  affected 
in  the  groin,  the  axilla,  or  the  neck ;  secondly,  the  septiccemic  form 
in  which  the  bacillus  reaches  the  blood ;  thirdly,  the  pneumonic,  in 
which  the  lungs  are  mainly  affected ;  and  fourthly,  pestis  minor,  in 
which  the  affection  of  the  glands  stops  short  of  the  septicsemic  stage, 
and  even  the  local  symptoms  are  slight.  There  are  certain  symptoms 
common  to  all  forms  of  plague,  when  at  all  severe. 

Symptoms  of  Plague.— An  ordinary  attack  of  plague  usually 
begins  three  to  five  days  after  exposure  to  infection.  Such  attack  may 
develop  gradually,  but,  generally,  there  is  sudden  onset  with  much 
fever,  as  indicated  by  a  high  temperature,  rapid  pulse,  headache,  hot 


SYMPTOMS  OF  PLAGUE  389 

skin,  and  thirst.  The  eyes  are  injected  as  if  inflamed ;  the  expression, 
at  first  haggard,  anxious  and  frightened,  becomes  subsequently  vacant, 
listless,  and  dull ;  the  utterance  is  thick,  and  the  gait  unsteady  as  in 
one  under  the  influence  of  drink.  Mental  aberration  develops  quickly. 
There  is  frequently  a  marked  tendency  to  faint.  The  tongue  is  at  first 
covered  with  a  moist  white  fur  except  at  the  edges,  which  are  red, 
but  later  on  it  becomes  dry  and  of  a  mahogany  colour.  Vomiting  and 
nausea  are  present  from  the  onset.  Sleeplessness  is  a  characteristic 
symptom. 

The  most  distinctive  sign  of  plague  is  the  presence  of  swellings, 
or  "  buboes  "  as  they  are  called,  in  the  groin,  armpit,  or  neck.  These 
"buboes,"  which  led  to  the  disease  being  called  "bubonic  plague," 
and  which  have  no  relation  to  venereal  complaints,  appear  as  a  rule 
on  the  second  or  third  day  of  the  disease.  They  occur  as  large, 
smooth,  tense  swellings.  They  are  usually  painful  and  tender  on 
pressure,  and  in  size  they  vary  from  that  of  an  almond  to  that  of 
an  orange.  Later  on  they  may  "  gather  "  and  burst  like  an  ordinary 
abscess.  There  may  appear  about  the  body  purple  spots,  and  even 
"  carbuncles." 

But  buboes  are  not  an  essential  feature  of  plague.  Cases  occur 
in  which  these  manifestations  of  the  disease  are  greatly  delayed  or 
even  absent,  as,  for  instance,  in  "  pneumonic,"  "  gastric,"  and  "  septi- 
csemic"  plague;  forms  of  the  malady  which  may  be  mistaken  re- 
spectively for  inflammation  of  the  lungs,  typhoid  fever,  or  acute 
blood  poisoning.  Plague  in  these  forms  is  always  grave ;  not  only 
because  of  the  fatality  of  the  cases  but  for  the  reason  that  they, 
especially  the  "pneumonic,"  are  highly  infectious  to  other  persons. 
It  is  important,  therefore,  that  in  localities  where  plague  is  present 
or  is  threatened,  cases  of  anomalous  illness  of  the  above  sorts  be 
without  loss  of  time  brought  under  medical  supervision. 

Besides  the  forms  of  plague  already  referred  to,  there  is  yet 
another,  namely,  the  so-called  "  ambulant  form."  In  plague  of  this 
description  the  affected  person  is  hardly  ill  at  all,  presenting  no  definite 
symptoms  perhaps  beyond  indolent,  though  painful,  swellings  in 
groin  or  armpit.  Such  plague  cases  may  nevertheless  be  instrumental 
in  spreading  the  disease,  and  any  persons  therefore  who,  having 
been  possibly  exposed  to  plague,  exhibit  these  symptoms,  should  be 
isolated  and  watched  medically  until  the  nature  of  their  malady  has 
been  definitely  ascertained. 

The  sudden  onset,  the  marked  prostration,  the  mental  aberration, 
the  splitting  headache,  vomiting  and  nausea,  backache,  the  rise  in 
temperature,  the  furred  tongue,  when  taken  in  conjunction  with 
tenderness  and  pain  in  some  one  of  the  groups  of  glands,  are  sufficient 
to  arouse  suspicion  as  to  the  case  being  one  of  plague. 

The  distribution  of  plague  at  the  present  time  is  fortunately  a 


390  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

somewhat  limited  one,  namely,  a  definite  area  in  Asia  known  as  the 
"  Plague  Belt."  From  Mesopotamia,  as  a  sort  of  focus,  the  disease 
spreads  northwards  to  the  Caspian  Sea,  westwards  to  the  Eed  Sea, 
southwards  as  far  as  Central  India,  and  eastwards  as  far  as  the 
China  Sea.  This  constitutes  the  "belt,"  but  the  disease  may  take 
an  epidemic  form,  and  is  readily,  though  very  slowly,  conveyed  by 
infection  or  contagion.  It  appears  to  be  infectious  by  means  of 
infective  dust,  and  contagious  by  prolonged  and  intimate  contact 
with  the  plague-stricken. 

Rats  and  Plague.. — Eats  have  been  shown  to  be  the  agents 
for  conveying  the  disease  from  port  to  port,  and  even  infecting 
man.  It  is  probable  that  rats  are  not  the  only  agency  acting 
in  this  way.*  Nevertheless,  it  is  true  that  rats  contract  the 
disease  more  readily  than  any  other  animals,  and  that  when 
suffering  from  it  they  may  spread  the  infection.  How  it  is 
thus  spread  is  not  known.  Cantlie  and  Yersin  have  pointed 
out  that  previously  to  an  epidemic  of  plague  rats  die  in  enormous 
numbers,  and  Manson  has  declared  that  rats  supply  "  the  best  and 
most  probably  the  initial  opportunity"  for  the  bacillus  of  plague. 
"  Were  I  asked,"  he  continues,  "  how  I  would  protect  a  state  from 
plague,  I  would  certainly  answer,  exterminate  the  rats  as  a  first  and 
most  important  measure."  But,  to  be  effective,  this  measure  must  be 
employed  in  anticipation  of  the  advent  of  the  disease.  "  When  the  rats 
are  tumbling  about  drunk  with  plague  it  is  too  late."  We  may  quote 
Sir  P.  Hanson's  simile  of  the  position  of  the  rat  in  epidemic  plague. 
"  I  would  compare  a  plague-threatened,  but  as  yet  not  invaded,  city," 
he  says,  "  to  a  grate  in  which  the  fire  is  laid  all  ready  for  lighting. 
There  is  the  refractory  though  combustible  coal  on  top,  there  is  the 
greasy  paper  and  dry,  resinous,  inflammable  wood  underneath,  and 
there  is  the  lighted  match  ready  to  be  employed.  Drop  the  match 
on  the  top  of  the  coal ;  it  flickers  for  a  second  and  goes  out — the 
coals  do  not  catch  fire.  But  apply  it  to  the  paper  and  sticks  under- 
neath, and  in  a  moment  there  is  a  blaze :  the  sticks  are  consumed, 
the  coals  catch,  and  in  a  little  while  the  fire  burns  merrily.  The 
coals  will  now  burn  by  themselves,  or,  if  they  threaten  to  go  out, 
another  stick  or  two  will  quickly  revive  the  fire.  In  my  simile  the 
coals  stand  for  the  human  inhabitants,  the  sticks  for  the  rodent 
inhabitants,  and  the  lighted  match  for  the  plague  germ  that  has 
dodged  the  quarantine  intended  to  protect  that  threatened  city.  NY> 
sticks,  no  fire ;  no  rats,  no  plague  epidemic."  f 

Dr  Doriga,  the  Principal  Medical  Officer  of  Health  for  Venice, 
has  set  forth  a  brief  rtsumt  of  the  chief  facts  relating  to  the 

*  See  Indian  Plaque,  Commission  Report,  1902,  and  Report  on  Playiifi  at  tfi/<hiey, 
1 903  (Ashburton  Thompson). 

t  Brit.  Med.  Jour.,  1899,  vol.  ii.,  p.  924. 


HATS  AND  PLAGUE  391 

agency  of  rats  and  mice  in  the  spread  of  plague,  which  is  as 
follows : — * 

"  1.  Kitasato  and  Yersin,  and  many  others  after  them,  have  found 
the  specific  bacillus  of  plague  in  the  dead  bodies  of  rats  and  mice 
collected  in  houses  in  which  cases  of  the  disease  subsequently  broke 
out  among  the  occupants,  or  in  the  streets  of  infected  towns.  They 
have  also  placed  beyond  question  the  great  susceptibility  of  these 
rodents  to  the  bacillus. 

"  2.  In  all  the-  towns  of  India  manifest  examples  of  contagion 
from  mice  to  men  have  been  observed.  At  Bombay,  in  certain 
establishments  where  the  dead  bodies  of  rats  were  found,  it  has  been 
noticed  that  the  persons  who  collected  them  alone  contracted  plague, 
although  many  other  work-people  were  engaged  at  the  same  place. 

"3.  The  first  cases  of  the  disease  have  sometimes  appeared  in 
warehouses  where  wheat,  cotton  seed,  or  other  substances  likely  to 
attract  rats  were  stored.  At  Kurachee,  where  the  warehouses  are 
situated  in  streets  without  dwelling-houses,  the  first  sufferers  were 
the  caretakers. 

"  4.  Well-constructed  and  well-maintained  houses,  i.e.  where  rats 
cannot  find  harbour,  nearly  always  remain  free  from  plague.  This 
same  immunity  was  demonstrated  by  Eennie  at  Canton,  in  1894, 
among  the  occupants  of  boats  anchored  in  the  river.  On  the  other 
hand,  is  to  be  observed  the  permanence  of  infection  in  the  houses  of 
poor  natives,  notwithstanding  the  removal  of  the  residents  and 
furniture  and  the  most  rigorous  disinfection,  because  of  reinfection 
by  means  of  mice. 

"5.  The  epidemics  at  Bombay,  Kurachee,  and  Karad  were 
chiefly  localised  in  quarters  where  the  disease  had  broken  out 
amongst  rats.  The  spread  of  infection  in  other  parts  of  these  same 
towns  was  regularly  preceded  by  the  immigration  and  death  of  rats, 
and  its  diffusion  always  corresponded  to  the  route  of  travel  taken  by 
these  rodents  in  their  migrations. 

"  6.  In  healthy  countries  adjoining  infected,  the  disease  broke  out 
amongst  the  inhabitants  without  the  importation  of  a  single  (human) 
case,  but  was  preceded  by  the  immigration  of  rats  from  an  infected 
place. 

"  7.  In  many  countries  and  towns  the  development  of  the 
epidemic  among  the  inhabitants  followed  a  month  after  the  importa- 
tation  of  the  first  cases,  or  after  the  death  of  fugitives  arriving  from 
infected  localities.  During  the  interval  the  plague  had  been  propa- 
gated by  mice. 

"  8.  Lastly,  the  mode  of  infection  and  propagation  of  plague  on 
certain  ships  proved  that  the  rats  on  board  had  been  the  vehicles  of 

*  "  The  Prevention  of  Plague  through  Suppression  of  Rats,"  Revue  d* Hygiene, 
August  1899. 


392  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

contagion."  *    It  should  be  added  that  Donga's  views  are  not  universally 
held,  and  were  not  fully  accepted  by  the  Indian  Plague  Commission. 

Quite  recently,  attempts  have  been  made  in  Paris,  with  the  con- 
sent of  the  Prefect  of  the  Seine,  to  exterminate  rats  wholesale,  in 
order  to  protect  the  city  from  an  epidemic  of  plague.f 

The  Bacteriology  of  Plague  is  one  of  the  latest  additions  to  the 
science.  During  the  Hong  Kong  epidemic  in  1894,  Kitasato,  of 
Tokio,  demonstrated  the  cause  of  plague  to  be  a  bacillus.  This  was 
immediately  confirmed  by  Yersin,  and  further  proved  by  the  isola- 
tion in  artificial  media  of  a  pure  culture  of  a  bacillus  able,  by  means 
of  inoculation,  to  produce  the  specific  disease  of  bubonic  plague. 

The  bacillus  was  first  detected  in  the  blood  of  patients  suffering 
from  the  disease.  It  takes  the  form  of  a  small,  round -ended,  oval 
cell  (O'V  /UL  broad  by  1*5  /m  long),  with  marked  polar  staining,  and 
hence  having  an  appearance  not  unlike  a  diplococcus.  In  the  middle 
there  is  a  clear  interspace,  and  the  whole  is  surrounded  with  a  thick 
capsule,  stained  only  with  difficulty.  The  organisms  are  often 
linked  together  in  pairs  or  even  chains  (especially  in  fluid  cultures), 
and  exhibit  polymorphic  forms.  In  culture  the  bacillus  may  be 
coccal  or  bacillary  in  form.  Involution  forms  occur  in  old  cultures, 
and  also,  more  rapidly,  when  2-5  per  cent,  of  sodium  chloride  is 
added  to  the  medium.  On  such  salt-agar  the  involution  forms  are 
very  marked.  The  bacillus  is  non-motile  (Plate  30,  p.  398). 

The  plague  bacillus  grows  readily  on  the  ordinary  media  at  blood- 
heat,  producing  smooth,  shining,  circular  cream-coloured  colonies, 
with  a  wavy  outline,  which  eventually  coalesce  to  form  a  greyish 
film.  The  colonies  slip  about  on  the  agar  when  touched  with  the 
platinum  wire.  If  melted  butter  (or  ghee)  or  oil  be  added  to 
bouillon,  this  bacillus  grows  in  "  stalactite  "  form,  that  is,  the  growth 
starts  on  the  under  surface  of  the  fat  globules,  and  extends  down- 
wards in  the  form  of  pendulous  string-like  masses  which  readily 
break  off  if  the  tube  is  slightly  shaken.  The  following  negative 
characters  help  to  distinguish  the  bacillus :  There  is  no  growth  011 
potato ;  milk  is  not  coagulated ;  gelatine  is  not  liquefied ;  Gram's 
method  does  not  stain  the  bacillus ;  and  there  are  no  spores.  The 
bacillus  is  readily  killed  by  heat  or  by  desiccation  over  sulphuric 
acid  at  30°  C.  Both  in  cultures  and  outside  the  body  the  bacillus 
loses  virulence.  To  this  may  be  attributed  possibly  the  variety  of 
forms  of  the  plague  bacillus  which  differ  in  virulence.  But  it  has 
great  powers  of  resistance  against  cold.]: 

On  gaining  entrance  to  the  human  body  the  bacillus  affects  in 

*  For  a  general  discussion  of  the  subject  of  plague  in  the  lower  animals,  see  Brit. 
Med.  Jour.,  1900,  i.  pp.  1141  and  1216.  ' 

t  Brit.  Med.  Jour.,  1900,  vol.  i.,  p.  722. 

£  For  further  particulars  as  to  cultured  characters  of  B.  peslis,  see  Brit.  Mad  Jour. , 
1902,  ii.,  95 6. 


THE  PLAGUE  BACILLUS  393 

particular  two  organs,  the  spleen  and  the  lymph  glands  (bubonic 
plague).  The  latter  become  inflamed  in  groups,  commencing  gener- 
ally with  the  inguinal  (60  per  cent.)  followed  by  the  axillary  (17  per 
cent.).  The  buboes  consist  usually  of  masses  of  inflamed  and  enlarged 
lymph  glands,  attended  with  haemorrhage  and  often  with  necrotic 
softening.  The  spleen  suffers  from  inflammatory  swelling,  which 
may  affect  other  organs  also.  In  both  places  the  bacilli  occur  in 
enormous  numbers.  In  the  pulmonary  form  the  lung  is  affected  with 
broncho-pneumonia.  This  form  of  plague  is  said  to  be  always  fatal. 
Kitasato  considers  that  the  bacillus  may  enter  the  body  by  the  three 
channels  adopted  by  anthrax,  namely,  (a)  the  skin,  (b)  alimentary 
canal,  and  (c)  respiratory  tract.  But  the  vast  majority  of  cases  arise 
from  infection  through  the  skin.  Infection  through  the  alimentary 
canal  is  still  doubtful.  Soil,  clothes,  and  contaminated  articles  gener- 
ally are  the  agencies  of  infection.  As  stated  already,  rats  play  an 
important  part  in  the  propagation  of  the  disease.  The  Indian  Com- 
mission hold  that  suctorial  insects  are  practically  of  no  importance 
as  transmitters  of  infection. 

Haffkine  has  prepared  a  vaccine  to  be  used  as  a  prophylactic  (see 
p.  424),  and  the  Indian  Plague  Commissioners  have  recently  reported 
on  its  effects.  Inoculation  with  this  vaccine  appears  sensibly  to 
diminish  the  incidence  of  the  plague  attacks  on  the  inoculated  popu- 
lation, although  the  degree  of  protection  is  not  perfect.  The  disease 
is  four  times  more  numerous  among  the  uninoculated  than  among 
the  inoculated.  The  fatality  of  the  attacks  is  also  diminished  in  the 
inoculated.  Protection  does  not  begin  till  a  few  days  after  the 
inoculation,  but  it  lasts  many  weeks  and  even  months.  It  may  here 
be  added  that  the  means  of  stamping  out  plague  are  the  ordinary 
methods  of  notification,  isolation,  and  disinfection.  The  latter  should 
include  destruction  of  the  patient's  clothes,  and  the  scraping  of  the 
walls,  and,  in  India,  burning  of  the  earthen  floor  of  his  dwelling. 
The  soil  and  dwellings  are  among  the  chief  sources  of  infection, 
and  therefore  require  most  attention. 

As  to  the  infectivity  of  plague,  it  is  now  generally  held  that  the 
bubonic  form  is,  as  a  rule,  dangerous  from  the  excretions,  and  only 
in  the  last  stages  of  the  disease ;  that  the  primary  pneumonic  form 
is  highly  infective ;  that  houses  in  which  plague  patients  or  plague 
rats  have  died,  and  in  which  clothing  has  been  soiled  by  excretions, 
are  infective ;  and  that  there  is  much  more  danger  from  living  in  an 
infected  house  than  from  coming  into  contact  with  a  plague  patient. 
Plague  is  a  disease  which  is  specially  favoured  by  insanitation  within 
the  walls  of  houses  as  contrasted  with  insanitation  outside  houses, 
relating,  for  example,  to  drainage,  removal  of  refuse,  etc.  Eats, 
merchandise,  clothing,  etc.,  may  each  and  all  play  a  part  in  the  con- 
veyance of  plague  from  one  village  to  another,  or  one  country  to 


394  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

another.  But  the  chief  agency  for  spreading  the  disease  to  unin- 
fected  places  consists  of  travellers.  The  lines  of  human  communica- 
tion are  followed  by  the  infection  in  a  marked  degree,  especially  lines 
of  steamship  and  railway. 

Plague  is  essentially  a  "  filth  disease,"  and  it  is  frequently  pre- 
ceded by  famine.  Temperature  and  overcrowding  exert  an  influence 
upon  it.  The  areas  affected  by  the  disease  in  the  Middle  Ages  in  the 
seventeenth  century,  and  in  1894-96,  are  alike  in  being  characterised 
by  filth  and  overcrowding.  There  is  little  fear,  speaking  generally, 
of  the  plague  ever  flourishing  under  Western  civilisation,  where  the 
conditions  are  such  that  even  when  it  appears  there  is  little  to 
encourage  or  favour  its  development* 

Administrative  Considerations.  —  Plague  will  not  readily 
fasten  on  any  section  of  a  population  which  is  properly  housed, 
cleanly,  and  generally,  in  a  sanitary  sense,  well-to-do  ;  rather  it  will 
especially  affect,  if  it  obtains  foothold  in  a  district,  insanitary  areas 
such  as  are  peopled  by  the  poorest  class,  and  where  overcrowding  of 
persons  in  houses  and  dirt  and  squalor  of  dwellings  and  of  inhabi- 
tants tend  to  prevail. 

In  these  circumstances,  and  from  an  administrative  point  of 
view,  the  following  facts  respecting  plague  should  be  borne  in 
mind : — 

(1)  Plague  has  an  incubation  period  of  three  to  five  (in  excep- 
tional cases  of  perhaps  eight  to  ten)  days. 

(2)  Plague  is  wont,  especially  in  its  earlier  manifestations,  to 
assume   a   mild   form,   or   even   to   present   anomalous   symptoms, 
tending  to  confound  it  with  other  and  more  innocent  diseases. 

(3)  Plague  in  all  its  forms  must  needs  be  regarded  as  personally 
infective. 

(4)  Plague  affects  rats  as  well  as  the  human  subject ;  it  may, 
indeed,  be  found,  causing  mortality  among  these  lower  animals  ante- 
cedent to  its  definite  invasion  of  the  population.     There  can  be  no 
doubt   that  the   rat  and  man  are,  as   regards   plague,  reciprocally 
infective. 

Although  Local  Authorities  should  be  on  their  guard  against 
plague,  when  cases  occur  at  the  ports  or  elsewhere  in  these  islands, 
it  is  not  intended  to  suggest  that  there  exists,  under  these  circum- 
stances, cause  for  alarm.  There  can  be  no  doubt  that,  in  this 
country,  hygienic  conditions  and  methods  of  dealing  with  infectious 
diseases  are  far  in  advance  of  those  of  former  centuries  wherein 
plague  was  repeatedly  epidemic  in  our  populations;  they  are  in 
advance,  too,  of  those  in  localities  abroad,  where  plague  has  shown 
itself  formidable  in  recent  years.  During  the  past  fifty  years  there 

*  The  most  complete  account  of  plague  hitherto  published  is  The  Report  of  the 
Indian  Playue  Commission,  1902,  vols.  i.-viii.  (see  in  particular  vol.  v.). 


THE  CONTROL  OF  PLAGUE  395 

has  occurred  in  England  and  Wales  a  large  diminution  in  the 
mortality  from  most  diseases  of  the  infections  class,  and  in  the  same 
period  typhus  fever  has  declined  almost  to  extinction.  This  latter 
disease  is  that  which,  as  regards  the  conditions  under  which  it 
becomes  prevalent,  most  closely  resembles  plague.  Wherefore  it 
may  be  confidently  anticipated  that  the  measures  of  sanitary 
improvement,  of  isolation  and  .of  disinfection,  which  have  been 
found  effectual  against  indigenous  disease  such  as  typhus,  will,  if 
promptly  and  thoroughly  brought  to  bear,  be  equally  effectual 
against  plague. 

First  among  measures  requisite  for  control  of  plague  is  prompt 
notification  to  the  local  authority  of  all  cases  of  the  disease 
occurring  in  their  district.  As  a  rule,  the  first  cases  of  an  outbreak 
will  require  bacteriological  diagnosis  in  addition  to  or  auxiliary  to 
clinical  diagnosis. 

Secondly,  in  the  event  of  plague  being  detected  in  any  district, 
the  measures  to  be  taken  to  prevent  its  spread  are,  generally 
speaking,  those  which  are  available  against  the  more  ordinary 
epidemic  diseases.  These  measures  include  prompt  removal  of  the 
sick  persons  to  hospital  and  their  isolation  therein ;  the  destruction 
or  thorough  disinfection  of  all  infected  articles,  with  the  effectual 
disinfection  also  of  the  invaded  dwelling-place ;  the  keeping  under 
observation  during  ten  days  after  detection  of  each  plague  case  all 
persons  who  have  been  in  contact  with  the  patient ;  house  to  house 
visitation  for  the  discovery  of  unreported  or  suspicious  cases;  the 
abatement  as  speedily  as  possible  of  all  insanitary  conditions  in  the 
locality  which  may  tend  to  the  spread  of  the  disease ;  and,  in  the 
case  of  death,  the  prompt  disposal  of  the  body,  with  all  due  precau- 
tions against  its  becoming  a  source  of  infection. 

Thirdly,  an  essential  measure  of  precaution,  in  view  of  the 
observed  relation  between  plague  in  rats  and  plague  in  the  human 
subject,  will  be  the  prompt  destruction  of  all  rats  in  districts 
threatened  or  invaded  by  plague,  care  being  taken  that  their 
carcases  are  collected  and  burnt  without  being  unduly  handled.* 

When  treated  in  a  well-appointed  hospital,  with  plentiful  fresh 
air  and  proper  attention  to  cleanliness  and  disinfection,  plague,  except 
in  its  pneumonic  and  septicsemic  forms,  shows  but  small  infective 
power ;  and  that  therefore  doctors  and  nurses  in  attendance  on  the 
sick  run  but  little  risk  of  contracting  the  disease.  Nevertheless,  these 
and  other  persons  brought  into  close  relation  with  plague  may  be 
afforded  protection  against  infection  by  submitting  themselves  to 
protective  inoculation  ten  days  before  contact  with  plague  cases. 

*  Danysz  has  suggested  the  killing  of  rats  by  infecting  them  with  an  organism 
fatal  to  them,  Brit.  Mnl.  Jour.,  1904,  vol.  i.,  p.  947. 


396  THE  ETIOLOGY  OF  TROPICAL  DISEASES 


Directions  for  Obtaining  and  Forwarding  for  Bacterioscopic  Examina- 
tion Material  from  Suspected  Plague  Cases 

A. — From  the  Living  Person. 

1.  Clean  with  soap  and  water,  and  then  with  alcohol,  the  last  phalanx  of  either 
the  second  or  third  finger.     When  dry,  or  after  mopping  with  a  clean  cloth,  put  a 
piece  of  tape  round  the  proximal  end  of  the  last  phalanx,  so  as  to  cause  venous  con- 
gestion.     Prick  the  palmar  surface  of  this   phalanx  with   a  sterile  needle,   and 
immediately  take  up  the  exuding  blood  in  two  sterile  capillary  tubes  such  as  are 
used  for  collecting  vaccine  lymph.     These  tubes  when  charged  should  be  sealed  at 
both  ends. 

2.  When  there  is  a  discharging  bubo,  collect  fluid  therefrom  in  capillary  tubes,  as 
in  the  case  of  blood.     When  this  discharge  is  not  of  a  sufficiently  fluid  character  for 
collection  in  this  way,  place  some  of  it  in  a  small  glass-stoppered  phial,  previously 
well  washed  out  with  alcohol,  care  being  taken  that  no  alcohol  remains  in  the 
phial. 

3.  If  expectoration  be  obtainable,  collect  some  in  a  phial  in  the  manner  pre- 
scribed in  section  2. 

[In  blood,  discharge,  or  expectoration,  cover  glass  preparations  should  be  made 
and  stained  by  simple  stains,  and  by  Gram's  method.  The  plague  bacillus  does  not 
stain  by  Gram's  method.  Cocci,  streptococci,  and  diplococcus  pneumonia  do  stain 
by  Gram's  method.  Cultivations  and  inoculations  must  also  be  made.] 

B. — From  the  Dead  Body. 

1.  Cut  out  any  inflamed  lymph  gland,  together  with  some  of  its  surrounding 
tissue,  and  place  the  whole  in  a  wide-mouthed  glass-stoppered  bottle  previously 
well  washed  out  with  alcohol,  care  being  taken  that  no  alcohol  remains  in  the  bottle. 
The  bottle  should  have  the  stopper  well  secured  and  sealed. 

2.  Obtain  also  a  piece  of  the  spleen,  dealing  with  it  in  the  same  manner. 

All  suspected  plague  material  should  be  carefully  packed,  so  as  to  avoid  risk  of 
breakage. 

4.  Leprosy 

This  ancient  disease  is  said  to  have  existed  in  Egypt  3500 
B.C.,  and  was  comparatively  common  in  India,  China,  and  even  in 
parts  of  Europe  500  B.C.  We  know  it  has  existed  in  many  parts 
of  the  world  in  the  past,  in  which  regions  it  is  now  extinct.  Some 
of  the  earliest  notices  we  have  of  it  in  this  country  come  from 
Ireland,  and  date  back  to  the  fifth  and  sixth  centuries.  Even  at 
that  period  of  time  also  various  classical  descriptions  of  the  disease 
had  been  written  and  various  decrees  made  by  Church  councils  to 
protect  lepers  and  prevent  the  spread  of  the  disease,  which  was 
often  looked  upon  as  a  divine  visitation.  In  the  tenth  century 
leprosy  was  prevalent  in  England;  it  reached  its  zenith  in  the 
thirteenth  century,  or  possibly  a  little  earlier,  and  declined  from 
that  date  to  its  extinction  in  the  sixteenth.  But  even  two  hundred 
years  later  leprosy  was  endemic  in  the  Shetlands,  and  it  is  recorded 
that  in  1742  there  was  held  a  public  thanksgiving  in  Shetland  on 
account  of  the  disappearance  of  leprosy.  The  last  leper  living  in  the 
Shetlands  was  admitted  to  the  Edinburgh  Infirmary  in  1798. 


LEPROSY  397 

At  one  time  or  another  there  were  as  many  as  200  institutions 
in  the  British  Isles  for  the  more  or  less  exclusive  use  of  lepers. 
Many  of  these  establishments  were  of  an  ecclesiastical  or  municipal 
character,  and  owing  to  the  fact  that  diagnosis  was  not  accurately  or 
carefully  made,  it  is  certain  that  these  institutions  frequently  housed 
persons  suffering  from  diseases  other  than  leprosy.  Bury  St 
Edmunds,  Bristol,  Canterbury,  London,  Lynn,  Norwich,  Thetford, 
and  York  were  centres  for  lepers.  Burton  Lazars  and  Sherburn, 
in  Durham,  were  two  of  the  more  famous  leper  institutions. 

Elsewhere,  the  writer  has  furnished  the  evidence  obtainable  in 
support  of  the  view  that  true  leprosy  (elephantiasis  grcecorum)  was 
prevalent  in  England  in  the  Middle  Ages.*  It  existed  there  anterior 
to  the  crusades,  which  per  se  had  little  or  no  effect  in  spreading  the 
disease  in  England.  It  was  generally  supposed  from  the  eleventh 
century  that  leprosy  was  a  contagious  and  hereditary  disease,  and 
that  it  depended  upon  these  two  characters  for  its  extension  in 
England.  But  probably  such  was  not  the  case,  for  it  is  fairly  certain 
that  strict  segregation  was  never  carried  out.  The  disease  as  an 
endemic  disease  reached  its  zenith  in  the  thirteenth  century  or 
earlier,  and  declined  till  final  extinction  in  the  eighteenth.  In 
England  itself  it  disappeared  approximately  in  the  sixteenth  century. 
Probably  the  famine  of  1315,  and  the  Black  Death  of  1349,  materi- 
ally assisted  in  the  extermination  of  lepers.  The  disease  being 
diffused  neither  by  contagion  nor  heredity  has  under  favourable 
hygienic  circumstances  a  tendency  to  die  out.  Hence  the  decline 
and  final  extinction  of  leprosy  in  Great  Britain  was  due  to  this 
general  tendency  under  favouring  circumstances,  viz.,  to  an  extensive 
social  improvement  in  the  life  of  the  people,  to  a  complete  change  in 
the  poor  and  insufficient  diet,  and  to  general  sanitary  advancement. 

At  the  present  time  the  distribution  of  the  disease  is  mostly 
Asiatic.  Norway  contains  about  1200  lepers,  Spain  a  smaller 
number.  Scattered  through  Europe  are  perhaps  another  2000  to 
3000,  in  India  100,000,  and  a  number  in  Japan.  The  Cape 
possesses  a  famous  leper  hospital  on  Eobben  Island,  with  a 
number  of  patients.  The  disease  is  also  endemic  in  the  Sandwich 
Islands. 

Descriptions  of  the  pathological  varieties  of  leprosy  have  been 
very  diverse.  The  classification  now  generally  adopted  includes 
three  forms :  the  tuberculated,  the  anaesthetic,  or  (maculo-ansesthetic), 
and  the  mixed.  Lepra  tuberculosa  is  that  form  of  the  disease 
affecting  chiefly  the  skin,  and  resulting  in  a  nodular  tuberculated 
growth  or  a  diffuse  infiltration.  It  causes  great  disfigurement.  The 
anaesthetic  form  causes  a  destruction  of  the  nerve  fibres,  and  so 

*  The  Decline  and  Extinction  of  Endemic  Leprosy  in  the  British  Islands,  1895, 
p.  108. 


398  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

produces  anaesthesia,  paralysis,  and  what  are  called  "  trophic " 
changes.  Not  infrequently  patches  occur  on  the  skin,  which  appear 
like  parchment,  owing  to  this  trophic  change.  Bullse  may  arise. 
When  the  tissue  change  is  radical  or  far  advanced,  considerable 
distortion  may  result.  The  mixed  variety  of  leprosy,  as  its  name 
implies,  is  a  mixture  of  the  two  other  forms. 

The  Bacteriology  of  Leprosy.— The  B.  leprce  was  discovered 
by  Hansen  in  1874.  He  found  it  in  the  lepra  cells  in  the  skin, 
lymph  glands,  liver,  spleen,  and  thickened  parts  of  the  nerves.  It 
is  common  in  the  discharge  from  the  wounds  of  lepers.  It  is 
conveyed  in  the  body  by  the  lymph  stream,  and  has  rarely  been 
isolated  from  the  blood  (Kobner). 

The  bacillus  is  present  in  enormous  numbers  in  the  skin  and 
tissues,  and  has  a  form  very  similar  indeed  to  B.  tuberculosis.  It  is 
a  straight  rod,  and  showing  with  some  staining  methods  marked 
beading,  but  with  others  no  beading  at  all.  It  measures  4  /m  long 
and  1  fj.  broad.  Young  leprosy  bacilli  are  said  to  be  motile,  but  old 
ones  are  not.  Neisser  has  maintained  that  the  bacillus  possesses  a 
capsule  and  spores.  The  latter  have  not  been  seen,  but  Neisser  holds 
that  this  is  the  form  in  which  the  bacillus  gains  entrance  to  the 
body.  There  is  a  characteristic  which  fortunately  aids  us  in  the 
diagnosis  of  this  disease  in  the  tissues,  and  that  is  the  arrangement 
of  the  bacilli,  which  are  rarely  scattered  or  isolated,  as  in  tubercle, 
but  gathered  together  in  clumps  and  colonies.  The  bacilli  occur 
for  the  most  part  inside  the  round  cells,  but  they  are  also  found  free 
in  the  lymphatics,  inside  connective  tissue  cells,  and  in  the  walls  of 
blood-vessels.  A  few  may  often  be  found  in  the  hair  follicles  or 
glands  of  the  skin,  or  even  in  the  epithelium.  The  bacilli  also  occur 
in  the  lymphatic  glands  and  in  the  internal  organs.  The  brain  and 
spinal  cord  are  almost  always  exempt.  But  recent  research  has 
made  it  evident  that  the  distribution  in  the  tissues  may  be  more 
widespread  than  was  formerly  supposed.  Bordoni  -  Uffreduzzi, 
Carrasquilla  (1899)  and  Campana,  claim  to  have  isolated  the  bacillus 
and  grown  it  on  artificial  media,  the  two  former  aerobically  on 
peptone-glycerine  blood  serum,  at  37°  C.,  the  latter  anaerobically. 
Other  workers  have  been  unable  to  obtain  successful  results.  Culti- 
vated bacteria  from  the  organs  of  lepers,  described  rather  later  by 
Babes,  and  still  more  recently  by  Czaplewski,  differ  from  the  genuine 
bacillus  of  leprosy  in  their  incomplete  resistance  to  acids.  Both 
authors  maintain  that  the  bacteria  cultivated  by  them  resemble  the 
bacilli  of  diphtheria.  In  any  case  it  is  very  doubtful  whether  these 
bacteria  cultivated  from  leprosy  are  the  genuine  ^Bacillus  hprcc. 
Hence  it  is  not  possible  to  study  the  bacteriology  of  leprosy  at  all 
completely ;  inoculation  experiments  also  have  not  proved  successful. 
Nevertheless  there  is  little  doubt  that  leprosy  is  a  bacterial  disease 


PLATE  30. 


mm. 


i 


Bacillus  leprce. 

Discharge  from  sore  of  leper.    Stained  by 

Ziehl-Neelsen  method. 

x   1000. 


Bacillus  leprce. 

Lepra  cells  containing  bacilli.     From  lobule  of  ear  of  leper. 

Stained  by  Ziehl-Neelsen  method. 

X  1000. 


Bacillus  of  Plague  (B.  pestis  bubonicce). 

From  liver  of  plague-stricken  rat. 

x  1000. 


Staphylococcus  pyogenes  aureus. 
x  1000. 


[To  face  page  398. 


LEPROSY  399 

produced  by  the  bacillus  of  Hansen.  Bordoni-  Uffreduzzi  maintains 
that  the  parasitic  existence  of  the  B.  leprce  may  alternate  with  a 
saprophytic  stage.  This  may  be  of  importance  in  the  spread  of  the 
disease.  There  is  evidence  in  support  of  the  non-communicability  of 
the  disease  by  heredity  or  contagion.  Segregation  does  not  appeal- 
always  to  result  in  a  decline  of  the  disease,  as  we  should  expect  if 
it  were  purely  contagious.  Ehlers,  of  Copenhagen,  has,  however, 
as  recently  as  1897  reaffirmed  his  belief  in  the  contagiousness  of 
leprosy ;  Virchow,  on  the  other  hand,  declared  that  it  was  not  highly 
contagious.  There  is  evidence  to  show  that  persons  far  advanced 
in  the  disease  may  live  in  a  healthy  community,  and  yet  not  infect 
their  immediate  neighbours.  Indeed,  the  transmission  of  the  disease 
is  still  an  unsolved  problem.  Mr  Hutchinson  maintains  that  diet, 
particularly  uncooked  or  putrid  fish,  is  a  likely  channel.  Deficiency 
of  salt,  telluric  and  climatic  conditions,  racial  tendencies,  social 
status,  poverty,  insanitation,  drinking-water,  even  vaccination,  have 
all  secured  support  from  various  seekers  after  the  true  channel  by 
which  the  bacillus  gains  entrance  to  the  human  body.  The  real 
mode  of  transmission  is,  however,  still  unknown.  The  decline  and 
final  extinction  of  leprosy  in  the  British  Islands  \p,s,  as  we  have 
stated,  probably  due  in  part  to  the  natural  tendency  of  the  disease 
to  die  out,  and  in  part  to  a  general  and  extensive  social  improvement 
in  the  life  of  the  people,  to  a  complete  change  in  the  poor  and 
insufficient  diet,  and  to  general  sanitation. 

At  the  Leprosy  Congress  held  in  Berlin  in  1897,  Hansen  again 
emphasised  his  belief  that  segregation  was  the  cause  of  the  decline 
of  leprosy  wherever  it  had  occurred.  But  there  appears  to  be 
evidence  to  show  that  leprosy  has  declined  where  there  has  been  no 
segregation  whatever,  and  therefore,  however  favourable  to  decline 
such  isolation  may  be,  it  would  seem  not  to  be  an  actually  necessary 
condition.  At  the  same  Congress  Besnier  declared  in  favour  of  the 
infective  virus  being  widely  propagated  by  means  of  the  nasal 
secretion.  Sticker  states  that  the  nasal  secretion  contains  myriads 
of  lepra  bacilli,  especially  in  the  acute  stages  of  the  disease,  and 
Besnier  and  Sticker  have  pointed  out  how  frequently  and  severely 
the  septum  nasi  and  skin  over  the  nose  is  affected  in  leprosy.  Several 
leprologists  in  India  have  recorded  similar  observations.  These  facts 
appear  to  support  Besnier's  contention,  that  the  disease  is  spread  by 
nasal  secretion. 

We  may  add  here  the  conclusions  arrived  at  by  the  English 
Leprosy  Commission  *  in  India : — 

"  1.  Leprosy  is  a  disease  sui* generis ;  it  is  not  a  form  of  syphilis 

*  Dated  1890-91.  The  Commissioners  were  the  late  Beaven  Rake,  M.D.,  G.  A. 
Buckmaster,  M.D.,  the  late  Prof.  Kanthack,  of  Cambridge,  the  late  Surgeon-Major 
Arthur  Barclay,  and  Surgeon-Major  S.  J.  Thomson. 


400  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

or  tuberculosis,  but  has  striking  etiological  analogies  with  the 
latter. 

"  2.  Leprosy  is  not  diffused  by  hereditary  transmission,  and,  for 
this  reason  and  the  established  amount  of  sterility  among  lepers,  the 
disease  has  a  natural  tendency  to  die  out. 

"  3.  Though  in  a  scientific  classification  of  diseases,  leprosy  must 
be  regarded  as  contagious,  and  also  inoculable,  yet  the  extent  to 
which  it  is  propagated  by  these  means  is  exceedingly  small. 

"  4.  Leprosy  is  not  directly  originated  by  the  use  of  any  particular 
article  of  food,  nor  by  any  climatic  or  telluric  conditions,  nor  by 
insanitary  surroundings,  neither  does  it  peculiarly  affect  any  race 
or  caste. 

"  5.  Leprosy  is  indirectly  influenced  by  insanitary  surroundings, 
such  as  poverty,  bad  food,  or  deficient  drainage  or  ventilation,  for 
these  by  causing  a  predisposition  increase  the  susceptibility  of  the 
individual  to  the  disease. 

"6.  Leprosy,  in  the  great  majority  of  cases,  originates  de  novo, 
that  is,  from  a  sequence  or  concurrence  of  causes  and  conditions  dealt 
with  in  the  Eeport,  and  which  are  related  to  each  other  in  ways  at 
present  imperfectly  known." 

The  practical  suggestions  of  the  Commission  for  preventive 
treatment  included  voluntary  isolation,  prohibition  of  the  sale  of 
articles  of  food  by  lepers,  leper  farms,  orphanages,  and  "improved 
sanitation  and  good  dietetic  conditions  "  generally.  Serum-therapy 
has  been  attempted  on  behalf  of  the  French  Academy  of  Medicine, 
but  without  success.  Many  forms  of  treatment  ameliorate  the 
miserable  condition  of  the  leper,  but  up  to  the  present  no  curative 
agent  has  been  found. 

5.  Yellow  Fever 

This  disease  is  admitted  to  be  one  of  the  most  terrible  of  tropical 
diseases.  Fortunately,  its  area  of  endemicity  is  comparatively 
limited.  When,  however,  it  breaks  out,  especially  on  board  ship, 
its  high  percentage  of  fatality  is  well  known. 

A  number  of  investigators,  from  the  beginning  of  last  century 
down  to  the  present  time,  have  been  at  work  on  the  cause  of  yellow 
fever.  Sanarelli,  the  Director  of  the  Institute  of  Hygiene,  in  the 
University  of  Montevideo,  in  South  America,  is  one  of  the  more 
recent  workers,  and  he  has  isolated  a  bacillus  which  he  believes  is 
the  causal  agent  of  the  disease.  The  bodies  of  those  who  die  of 
yellow  fever  are,  however,  either  so  free  from  organisms,  or  so  entirely 
invaded  by  organisms,  that  the  B.  icteroides  is  difficult  to  discover. 
Moreover,  led  by  the  clinical  signs  of  the  disease — "  black  vomit "  and 
other  gastro-intestinal  phenomena  —  investigators  have,  a  priori, 
supposed  that  the  digestive  canal  was  the  seat  of  the  disease,  and 


YELLOW  FEVER  401 

therefore  the  probable  locality  of  the  causal  bacillus;  whereas,  as 
Sanarelli  pointed  out,  the  B.  icteroides  must  be  sought  for  in  the  blood 
and  tissues,  and  not  in  the  alimentary  canal.  But  even  thus  the 
difficulties  are  not  wholly  removed.  For  it  happens  that  this 
organism  may  only  be  found  in  comparatively  small  numbers,  and 
certainly  at  the  beginning  of  the  disease  multiplies  very  little  in 
the  human  body.  Its  influence  upon  the  body,  too,  appears  to  be 
such  that  the  tissues  of  a  yellow  fever  patient  become  the  hunting 
ground  of  vast  numbers  of  secondary  infective  bacteria. 

This  bacillus  (B.  icteroides)  may  be  obtained  from  the  small 
capillaries — in,  say,  the  liver — by  incubation  at  favourable  tempera- 
ture (37°  C.).  It  is  a  short  bacillus  with  round  ends,  like  B.  coli.  It  is 
motile,  and  possesses  4-8  flagella.  It  develops  sufficiently  well  for  all 
practical  purposes  on  the  ordinary  media.  On  agar  at  blood-heat  it 
grows  well — a  grey,  iridescent,  smooth  layer,  with  regular  margins ; 
and  on  the  same  medium,  at  the  temperature  of  the  room,  it 
produces  in  twenty-four  hours  characteristic  colonies  not  unlike  drops 
of  milk.  It  grows  on  gelatine  without  liquefaction.  The  organism 
is  a  facultative  anaerobe,  decolorised  by  Gram's  method;  ferments 
sugar,  but  does  not  coagulate  milk  until  after  some  weeks.  It 
appears  strongly  to  resist  drying.  Direct  sunlight  kills  it  in  seven 
hours ;  but  it  is  said  to  be  able  to  live  for  some  time  in  sea  water. 
The  organism  can  be  isolated  from  the  living  patient  as  well  as  the 
dead  body.* 

Sanarelli  has  maintained  that  atmospheric  transmission  is  the 
common  channel  of  infection  in  yellow  fever.  As  everyone  knows, 
it  is  a  disease  which,  when  once  installed  on  board  ship,  seems  to 
cling  to  it  tenaciously,  more  particularly  in  the  hold,  magazines, 
merchandise,  and  in  all  close  and  restricted  quarters.  Humidity, 
heat,  and  want  of  light  and  ventilation  have  been,  until  recently,  the 
supposed  conditions  necessary  to  the  conveyance  or  harbouring  of 
yellow  fever.  Sanarelli  has  further  suggested  that  moulds  must  be 
considered  "the  natural  protectors  of  the  specific  agent  of  yellow 
fever."!  By  a  series  of  interesting  experiments,  he  demonstrated 
the  stimulating  effect  which  moulds  have  upon  gelatine-plate  cultures 
of  this  bacillus  in  the  laboratory.  Outside  the  laboratory,  in  houses 
and  on  ships,  the  conditions  favouring  the  growth  of  moulds  appeared 
also  to  be  the  conditions  favouring  yellow  fever.  For  instance, 
humidity,  heat,  and  scanty  aeration  are  highly  favourable  to  mould 
growth,  and  thus,  according  to  Sanarelli,  to  yellow  fever.  To  these 
factors,  also,  is  supposed  to  be  due  the  unhealthiness  of  Eio  Janeiro. 
During  the  yellow  fever  epidemic  in  Montevideo  in  1872,  the 

*  Brit.  Med.  Jour.,  1897,  vol.  ii.,  p.  7  (Prof.   G.   Sanarelli),  1900,  vol.  i.,  p.   334, 
and  The  Medical  News  (New  York),  9th  December  1899. 
t  Brit.  Med.  Jour.,  1897,  vol.  ii.,  p.  11. 

2  C 


402  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

inhabitants  of  the  houses  facing  north  were  attacked  much  more 
than  others,  and  it  was  found  that  both  these  houses  and  the  streets 
in  which  they  stood  were  distinguished  by  an  exceptional  degree  of 
humidity. 

In  1901  the  United  States  Army  Commission  reported,  after 
extensive  investigations  into  the  etiology  of  yellow  fever,  that  whilst 
B.  icteroides  was  not  always  present  in  cases  of  yellow  fever,  the 
blood  of  the  patient  appeared  to  contain  the  virus,  whatever  it  was, 
and  retained  it  after  being  passed  through  a  Berkefeld  filter.  The 
Commission  further  reported  that  the  disease  was  not  communicable 
by  direct  contact  with  those  suffering  from  the  disease,  but  was 
probably  communicated  by  mosquitoes  in  a  similar  way  to  malaria. 
The  species  of  mosquito  found  capable  of  carrying  the  infection  in 
this  way  is  the  Stegomyia  fasciata.  Though  the  matter  was  not 
proved,  nor  the  nature  of  the  virus  determined,  preventive  mea- 
sures were  adopted  in  Havana  on  the  mosquito  hypothesis,  with 
the  remarkable  result  that  the  disease  was  stamped  out. 
Guite*ras  of  Havana  has  carried  out  further  experiments  which 
confirm  many  of  the  Commission's  findings,  and,  in  particular,  the 
transmission  of  the  disease  by  mosquitoes.* 

In  1902  a  United  States  Army  Expedition  was  appointed  to 
reinvestigate  the  subject,  and  it  reported  in  1903.  The  chief 
conclusions  reached  were  as  follows  :  (1)  Bacteriological  examination 
of  the  blood  of  persons  with  uncomplicated  yellow  fever  during  life, 
as  well  as  of  organs  and  blood  immediately  after  death,  is  negative. 
(2)  The  mosquito  known  as  Stegomyia  fasciata,  when  allowed  to  suck 
the  blood  of  a  yellow  fever  patient  after  the  lapse  of  forty-one  hours 
after  the  onset  of  the  disease,  and  subsequently  fed  on  sugar  and 
water  for  twenty-two  days  can,  if  permitted  to  bite  a  non-immune 
person,  produce  a  severe  attack  of  the  disease.  (3)  Stegomyia  fasciata, 
contaminated  by  sucking  the  blood  of  a  yellow  fever  patient,  and 
then  killed,  cut  into  sections  and  appropriately  stained,  presents  with 
regularity  a  protozoan  parasite,  Myxo-coccidium  stegomyice,  which 
can  be  traced  through  a  cycle  of  developments  from  the  gamete  to 
the  sporozoite.  (4)  Stegomyia  fasciata,  fed  on  the  blood  of  a  person 
with  malarial  fever,  on  normal  blood,  or  artificially,  does  not  harbour 
the  myxo-coccidium. 

The  etiology  of  yellow  fever,  therefore,  remains  at  the  present 
time  sub  judice,  but  the  probabilities  are  that  the  disease  is  mosquito- 
borne  and  due  not  to  bacteria  but  to  sporozoal  parasites. 

There  are  other  tropical  diseases  to  which  brief  reference  must  be  made. 
Malta  Fever  (Mediterranean  fever)  is  common  along  the  coast  of  the  Mediter- 
ranean and  on  its  islands.     It  also  occurs  elsewhere.     In  1886  Bruce  cultivated  from 

*  American  Medicine,  23rd  November  1901,  p.  809. 


MALTA  FEVER,  SLEEPING  SICKNESS,  ETC.  403 

the  spleen  of  patients  dead  of  the  disease  an  organism  now  known  as  the  Micrococcus 
melitensis.  Clinically,  Malta  fever  is  a  disease  of  long  duration  and  variable 
symptoms,  including  remitting  fever.  Perspiration,  pains,  swelling  of  joints,  en- 
largement of  the  spleen,  etc.,  are  among  the  common  signs.  Micrococcus  melitensis 
is  a  small,  round,  or  slightly  oval  coccus,  singly,  in  pairs,  or  chains.  Does  not  stain 
by  Gram's  method.  Can  be  cultivated  on  agar  at  37°  C.  from  the  spleen ;  colonies 
appear  about  third  day  as  small,  round,  slightly  raised  growths,  old  cultures  assume 
a  buff  tint.  Addition  of  nutrose  hastens  growth  of  culture.  On  gelatine  growth  is 
very  slow ;  there  is  no  liquefaction.  In  broth  there  is  a  turbid  growth,  without 
pellicle  formation. 

Cultures  kept  at  22°  C.  retain  their  vitality  for  fourteen  months,  but  the  organism 
dies  in  about  five  days  in  sterile,  fresh,  and  sea  water  and  urine,  but  remains  active 
for  longer  in  sterile  milk,  and  for  sixty-nine  days  in  dust.  The  organism  is  present 
in  the  peripheral  blood  in  all  cases  during  the  early  stages,  and  in  severe  pyrexial 
relapses.  It  has  recently  been  isolated  from  the  urine  of  patients.  The  disease 
appears  to  be  inoculable  in  animals. 

Sleeping  Sickness.  — Investigations  point  to  the  conclusion  that  sleeping  sickness 
is  caused  by  the  entrance  into"  the  blood,  and  thence  into  the  cerebro-spinal  fluid,  of 
a  species  of  trypanosoma  (probably  the  Trypanosoma  gambiene,  discovered  by  Forde 
and  described  by  Button),  which  is  transmitted  from  the  sick  to  the  healthy  by  a 
species  of  tsetse  fly  (Olossina  palpalis),  and  by  it  alone ;  that,  in  short,  sleeping 
sickness  is  a  human  tsetse-fly  disease.  From  a  series  of  carefully  controlled  and 
minutely  observed  experiments,  carried  out  by  Bruce,  Nabarro,  and  Grieg,  it  was 
discovered  that  monkeys  inoculated  with  cerebro-spinal  fluid  from  sleeping  sickness 
patients,  or  with  blood  from  natives  not  as  yet  showing  symptoms  of  sleeping  sick- 
ness, but  containing  a  similar  parasite,  sickened  and  died  with  all  the  symptoms  of 
sleeping  sickness. 

From  the  analogy  of  the  closely  related  disease  in  cattle,  the  nagana  or  tsetse  fly 
disease  of  South  Africa,  it  was  suspected  that  in  sleeping  sickness  a  like  method  of 
infection  took  place.  It  has  been  demonstrated  by  experiment  that  not  only  were 
these  flies,  fed  on  sleeping  sickness  cases,  capable  of  conveying  the  disease  to  healthy 
monkeys,  but  that  the  freshly  caught  flies  from  an  infected  area,  without  any  arti- 
ficial feeding,  were  also  capable  of  conveying  the  disease. 

It  was  further  discovered  by  a  carefully-organised  investigation  that  this  fly,  like 
its  congener  the  tsetse  fly  of  South  Africa,  is  confined  to  well-defined  areas,  and  that 
these  areas  correspond  absolutely  with  the  distribution  of  sleeping  sickness  ;  whereas, 
in  regions  where  no  Glossina  palpalis  is  found,  although  other  biting  flies  abound, 
there  is  no  sleeping  sickness.  Moreover,  an  examination  of  a  large  number  of 
individuals  in  the  sleeping  sickness  areas  and  the  non-sleeping  sickness  areas 
respectively,  revealed  the  fact  that,  while  a  large  percentage  (28)  of  the  inhabitants  of 
the  sleeping  sickness  areas  have  in  their  blood  the  trypanosoma  already  referred  to, 
in  not  a  single  case  taken  from  inhabitants  of  non-sleeping  sickness  areas  was  this 
parasite  found."  The  only  other  human  trypanosome  at  present  known  is  that 
occurring  in  trypanosomiasis. 

Dysentery  is  another  tropical  disease  in  which  the  etiology  has  not  been  finally 
worked  out.  Endemic  or  tropical  dysentery  is  possibly  due  to  Amoeba  coli.  Epi- 
demic dysentery  is  more  probably  due  to  Bacillus  dysenteric,  and  sporadic  and 
parasitic  dysentery  is  due  to  various  parasites,  such  as  Balantidium  coli  and  the 
Bilharzia.  B.  dysenteries  is  a  short  rod,  often  occurring  in  pairs  ;  non-motile ;  does  not 
stain  by  Gram's  method ;  does  not  curdle  milk  nor  liquefy  gelatine,  f  The  researches 

*  See  also  Brit.  Med.  Jour.,  1903,  vol.  i.,  p.  1431;  and  vol.  ii.,  pp.  1343  and 
1427  ;  and  Lancet,  1904  (July),  p.  290,  for  a  resume. 

f  For  a  full  discussion  of  the  subject,  see  Brit.  Med.  Jour.,  1901,  vol.  ii.,  p.  786, 
"A  Comparative  Study  of  the  Bacilli  of  Dysentery";  and  1903,  vol.  i.,  p.  1315, 
"  Amoebic  Dysentery  in  India " ;  and  Report  of  Royal  Commission  on  Dysentery, 
1903.  For  description  of  B.  dysenteries,  see  Report  of  Medical  Officer  to  Local  Govt. 
Bd.,  1901-02,  p.  396;  Brit.  Med.  Jour.,  1904,  vol.  i.,  p.  1002;  Edin.  Med.  Jour. 
(June),  1904,  p.  489  (Eyre). 


404  THE  ETIOLOGY  OF  TROPICAL  DISEASES 

of  Shiga,  Kruse,  Flexner,  and  others  point  to  acute  dysentery  being  caused  by  the 
specific  bacillus  B.  dysenteric?,  or  some  member  of  that  group  of  organisms.  Mott 
holds  that  "  asylum'  dysentery "  is  identical  with  tropical  dysentery,  and  both 
conditions  are  in  all  probability  of  bacillary  origin." 

Beri-beri. — The  first  medical  writer  to  describe  beri-beri,  and  by  that  name,  was 
Dr  J.  D.  Malcolmson,  F.  R.  S. ,  of  the  Madras  Medical  Service,  in  a  paper  published 
in  1835.  Sir  Joseph  Fayrer,  F.R.S.,  wrote  on  it,  identifying  one  form  of  it  with 
the  barbiers  of  the  earlier  European  travellers,  t  The  disease  is  endemic  in  Western 
India,  in  the  Indian  Archipelago,  and  throughout  the  coasts  of  Further  India  and 
Upper  India,  or  China  and  Japan.  It  is  practically  confined  to  the  labouring  classes 
where  they  are  vegetarians.  Dr  Wallace  Taylor  traces  it  to  a  microscopic  spore 
infecting  rice ;  and  other  observers  consider  it  a  "place  disease."  The  salient  fact 
is  that  it  almost  exclusively  attacks  those  who  are  engaged  in  hard  labour  on  insuffi- 
cient nourishment,  and  it  may  be  denned  as  the  scurvy  of  the  tropics.  It  is  marked 
by  extreme  weakness  and  dropsical  distension  of  the  abdomen,  limbs,  and  face,  both 
symptoms  developing  so  rapidly  as  to  alarm  alike  the  sufferer  and  those  attending  to 
him.  Hence  its  name  beri,  meaning  "  debility,"  and  the  reduplication  of  it,  beri-beri 
signifying  "  extreme,"  **  alarming,"  "  fatal,"  debility. 

The  disease  is  in  all  probability  a  germ  disease  but  possibly  not  communicable 
from  man  to  man.  It  may  be  that  the  germ  resides  in  soil  or  rice,  or  houses,  and 
surroundings  of  beri-beri  localities,  and  produces  a  toxin  which  on  being  absorbed 
produces  a  disease  having  many  similarities  with  alcoholic  neuritis  (Manson).  This 
may  be  the  explanation  of  the  view  that  beri-beri  is  a  "  place  disease."  Pekelharing 
and  Winkler  hold  that  they  have  isolated  a  bacterium  which  is  the  cause  of  the 
disease,  but  their  views  nave  not  been  generally  accepted. 

*  See  also  Bacteriological  and  Clinical  Studies  of  the  Diarrhceal  Diseases  of  Infancy, 
by  Flexner  and  Emmett  Holt  (Rockefeller  Inst.  Rep.),  1904. 

t  Practitioner  (January),  1877 ;  see  also  Report  on  Prison  Administration  in 
Burma,  1878. 


CHAPTEK  XII 

THE   QUESTION   OF   IMMUNITY  AND   ANTITOXINS 

Bacterial  Products — Toxins— Question  of  Immunity — Kinds  of  Immunity — Theories 
of  Immunity — Applications  of  Immunity — Vaccination  for  Small-pox  :  Effect 
of  Vaccination — Pasteur's  Treatment  for  Rabies — Inoculations  for  Cholera, 
Typhoid,  and  Plague— Antitoxin  Treatment  of  Diphtheria  and  its  Effects. 

THE  term  natural  immunity  is  used  to  denote  natural  resistance  to 
some  particular  specific  disease.  It  may  be  due  to  species  of  animal, 
or  age,  or  individual  idiosyncrasies.  We  not  infrequently  meet  with 
examples  of  this  freedom  from  disease.  Certain  species  of  animals 
do  not,  as  a  rule,  take  certain  diseases.  For  example,  cholera  and 
typhoid,  which  affect  man,  do  not  affect  the  lower  animals.  Swine 
plague,  which  affects  swine,  does  not  affect  man.  The  white  rat 
is  immune  to  anthrax,  which  readily  attacks  cattle.  Such  examples 
might  easily  be  multiplied.  Children,  again,  are  susceptible  to 
certain  diseases  and  insusceptible  to  certain  others  to  which  older 
people  are  susceptible.  The  young  of  the  lower  animals  also  are 
susceptible  to  diseases  which  do  not  attack  adult  animals.  We 
know,  too,  that  some  individuals  have  a  marked  protection  against 
certain  diseases.  Some  persons  coming  in  the  way  of  infection  at 
once  fall  victims  to  the  disease,  whilst  others  appear  to  be  proof 
against  it. 

It  is  only  in  recent  times  that  any  intelligent  explanations  have 
been  offered  to  account  for  these  phenomena.  The  most  recent,  and 
that  which  appears  to  have  most  to  substantiate  it,  is  known  as 
immunity  due  to  antitoxins.  To  understand  the  nature  of  antitoxins 
it  is  necessary  to  consider  briefly  the  products  of  bacterial  activity. 
They  are  chiefly  seven  : — 

405 


406     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

1.  Pigment. — We  have  already  seen  that  many  organisms  exhibit 
their    energy    in   the   formation  of   various   pigments.     These   are, 
as  a  rule,  "  innocent "  bacteria.      Oxygen  is  required  for  the  pro- 
duction of  pigment  by  some  of  these  species,  absence  of  light  by 
others,  and   they  all   vary  according  to   the  medium   upon  which 
they  are  growing.     Eed  milk,  blue  milk,  and  green  pus  are  illus- 
trations of   materials  owing  their  colour  to  pigment  produced  by 
bacteria.      Chromoporous  bacteria  are  those  in  which  the  pigment  is 
diffused  out  into  the  surrounding  medium;  cJiromophorous  bacteria 
are  those  in  which  the  pigment  is  stored  in  the  cell  protoplasm  of 
the  organism. 

2.  Gas. — A  large  number  of  the  common  bacteria,  like  B.  coli, 
produce  gas  in  their  growth;    hydrogen  (H),  carbonic  acid  (C02), 
methane   (CH4),   sulphuretted  hydrogen   (H2S),  and   even   nitrogen 
(N)  being  formed  by  different  bacteria. 

3.  Acids. — Lactic,  acetic,  butyric,  etc.,  are  common  types  of  acids 
resulting  from  the  growth  of  bacteria. 

4.  Liquefying  Ferment. — As  we  have  seen,  bacteria  may  also  be 
classified  with  regard  to  their  behaviour  in  gelatine  medium,  as  to 
whether  or  not  they  produce  a  peptonising  ferment  which  liquefies 
the  gelatine. 

5.  Phosphorescence. — Some  species  of  bacteria,  for  example,  certain 
species  in  sea -water,  possess  the  power  of  producing  light  (photogenic 
bacteria). 

6.  Many  organisms  are  capable  of  producing  indol  (a  substance 
formed  by  bacterial  action  from  proteids  by  alimentary  decomposi- 
tion), or  other  metabolic  substances  as  end-products. 

7.  Organic  Chemical  Products. — When  a  pathogenic  bacillus  grows 
in  the  body,  it  produces  as  a  result  of  its  metabolism  certain  poisonous 
substances  termed  toxins.     These  may  occur  in  the  blood  as  a  direct 
result  of  the  life  of  the  bacillus,  or  they  may  occur  as  the  result  of 
a  ferment  produced  by  the  bacillus.     Toxins  are  of  various  kinds, 
and  by  their  effect  upon  the  blood  and  body  tissues  they  cause 
the   symptoms  of   the   various   diseases.     We   know,  for   instance, 
that  a  characteristic  symptom  common  to  many  diseases  is  fever, 
which  is  produced  by  the  action  of   the  albumoses  (bodies   allied 
to  the  albumins)  upon   the   heat-regulating  centres  in   the   brain. 
Whenever   we   have   a   bacillus   growing  in   the   body   which    has 
the  power  of   producing  a  toxin  albumose,  we  obtain  fever  as  a 
result  of   that   product   acting  upon   the   brain.     Albumoses,  as  a 
matter  of  fact,  cause  a  number  of  symptoms  and  poisonous  effects, 
but   the   mention   of   one   as   an   illustration  will  suffice,      Toxins 
act,   broadly   speaking,   in    two   ways.     They    have    a    local   effect 


ACTION,  OF,j  TOXINS  407 

and  a  specific  effect,  as  the  two  following  illustrations  will   make 
evident : — - 

(1)  They  have  a  local  action,  as,  for  example,  in  the  formation 
of  an  abscess.     The  presence  of  the  causal  bacteria  in  the  tissue 
brings  about  very  marked  changes.     There  is  a  multiplication  of 
connective  tissue  corpuscles,  an  emigration   of   leucocytic   cells,   a 
congestion  of  blood  corpuscles.     These  elements  contribute  towards 
creating   a   swelling  and   redness,  and  pain  results  owing   to   the 
subsequent  pressure  upon  the  nerve  endings.     We  have,  in  short, 
a  state  of   inflammation.      It   is   then   that   the   toxin   commences 
its  local  action.     The  oldest  cells  in  the  mass  of  congestion  will 
break  down,  and  necrosis  or   death  will  rapidly  set  in.     The  con- 
nective  tissue   cells,   leucocytes,   blood   corpuscles,   etc.,   will    thus 
lose  their  form  and  function,  and  become  pus.     The  local  breaking 
down   of   these  gatherings   of   cells   into   fluid   matter  is   believed 
to   be   the   work,   not   of    the    bacteria    themselves,   but    of    their 
toxins. 

(2)  Toxins  may  be  absorbed  and  distributed  generally  throughout 
the  body.     "When  this  occurs  they  produce  degenerative  changes  in 
muscles,  in   organs,   and  in   the   blood   itself.     Of   such   a   change 
diphtheria  is  an  example.     The  bacillus  occurs  in  a  false  membrane 
in   the   throat,  and   occasionally   other   parts.     It   first   causes   the 
inflammatory  condition  giving  rise  to  the  membrane,  and  then  it 
breaks  it  down.     In  the  body  of  the  membrane  the  bacillus  appears 
to  secrete  a  ferment  which  by  its  action  and  interaction  with  the 
body  cells  and  proteids,  chiefly  those  of  the  spleen,  produces  albumoses 
and  an  organic  acid  (Martin).     These  latter  bodies  are  the  toxins. 
They  are  absorbed,  and  pass  throughout  the  body.     As  a  result,  we 
get  the  frequent  pulse  and  high  temperature  of  fever:   the  toxins 
irritate  the  mucous  membrane  of  the  intestine,  and  cause  various 
fermentative  changes  in  the  contents  of  the  intestines,  therefore  we 
get  the  symptoms  of  diarrhoea :  they  penetrate  the  liver,  spleen,  and 
kidney,  setting  up  fatty  degeneration  and  its  results  in  these  organs : 
they  finally  affect  many  of  the  motor  and  sensory  nerves,  breaking 
up  their  axis  cylinders  into  globules,  and  producing  the  characteristic 
paralysis.     Loss  of  weight  naturally  follows  many  of  these  degenera- 
tive or  wasting  changes.     Thus,  then,  we  have  some  of  the  chief 
changes  set  up  by  the  toxins,  and  these  changes  constitute  the  leading 
symptoms  in  the  disease  as  it  is  known  clinically.     In  addition  to 
the  presence  of  the  specific  bacillus  in  the  membrane,  we  also  have 
a  number  of  other  organisms,  like  the  B.  coli,  Streptococcus  pyogenes, 
and  various  staphylococci,  diplococci,  etc.     Each  of  these  produces, 
or  endeavours  in  the  midst  of  keen  competition  and  strife  to  produce, 
its   own    specific    effect.      Thus   we   obtain    the    complications    of 
diphtheria,  such  as  various  suppurative  and  septic  conditions.     The 


408     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 


whole   of  this  compound    process    may   be   tabulated   roughly   as 
follows : — * 


Bacillus  coli. 
Staphylococci. 
Diplococci. 
Streptococci. 


Toxins. 


Suppurative  glands, 
septic  poisoning, 
etc. 


BACILLUS  or  DIPHTHERIA  =  primary  infective  agent. 
Inflammatory  changes  and  fibrinous  exudation. 
FERMENT  IN  MEMBRANE  =  secondary  infective  agent. 
Passes  through  body,  and 


| 

1.  Fever. 

2.  Diarrhoea. 

3.  Loss  of  body  weight. 

4.  Fatty  degeneration. 

5.  Degeneration  of  peri- 

pheral   nerves     and 
resulting  paralysis. 


Such  is  the  specific  effect  of  toxins  in  diphtheria.  The  same 
principles  apply  with  equal  force  in  tetanus,  typhoid,  etc.,  the 
differences  being  in  degree  of  virulence,  specificity,  mode  of  onset, 
and  portions  of  the  body  affected. 

Sidney  Martin  suggested  a  provisional  classification  of  bacterial 
toxins  as  follows  :  — 


—  Extracellular 
bacterial  poisons. 


1.  Poisons  secreted  by  the  bacterium  itself  =  (ferment  ? 

toxin?) 

2.  Products  of  digestive  action  of  bacterium  =  albumoses  ;  I 

3.  Final  non-proteid  products  =  animal  alkaloid  ; 

4.  Poisons  present  in  the  body  of  the  bacillus  { 

Such  occur,  for  example,  in  the  tubercle  bacillus  and  the  cholera  vibrio. 


The  toxins  of  bacteria  are  of  a  kind  which  cannot  be  fully 
expressed  chemically,  but  only  pathologically.  They  are  probably 
of  a  ferment  nature  in  diphtheria  and  tetanus.  The  arguments  in 
support  of  that  view  are — (1)  that  they  act  in  infinitesimal  doses ; 
(2)  that  they  may  act  slowly  and  produce  death  after  many  days  by 
profoundly  affecting  the  general  nutrition;  and  (3)  that  they  are 
sensitive  to  the  action  of  heat  in  a  way  that  no  chemical  poisons  are 
known  to  be.  If  they  are  considered  as  ferments,  they  must  be 

*  It  should  be  distinctly  understood  that  this  table  is  merely  schematic  and 
provisional.  The  details  of  toxin  production  and  its  effect  are  of  course  still  open 
to  revision  and  amendment. 

f  Sidney  Martin,  M.D.,  F.R.S.,  F.R.C.P.,  Croonian  Lectures  delivered  before  the 
Royal  College  of  Physicians,  June  1898. 


ACTION  OF  TOXINS  409 

substances  which  have  a  peculiar  affinity  for  certain  tissues  of  the 
body  on  which  they  produce  their  special  toxic  effect.  Hitherto,  all 
.attempts  at  the  separation  of  such  bacterial  ferments  have  been 
without  success,  and  for  other  reasons  also  the  whole  question  of 
such  ferments  must  be  left  open  at  present.  Sidney  Martin  and 
others  have  demonstrated  that  many  of  the  extra-cellular  toxins  are 
albumoses  or  bodies  of  a  similar  nature.  They  are  non-crystallisable, 
soluble  in  water,  precipitated  along  with  the  proteids  by  concentrated 
alcohol,  relatively  unstable,  having  their  toxicity  diminished  or 
destroyed  by  heat,  light,  or  certain  chemical  agents.  As  for  the 
products  of  digestion,  they  are  formed  either  by  the  bacillus  ingest- 
ing the  proteid  and  discharging  it  as  albumose,  or  the  digestion 
occurs  by  means  of  a  ferment  secreted  by  the  bacillus  in  the  body  of 
the  individual  or  animal  suffering  from  the  disease. 

It  is  now  held  by  some  that  the  virus  of  anthrax  produces 
albumoses  and  an  alkaloidal  substance  (Martin),  the  former  producing 
fever,  the  latter  oedema,  congestion,  and  local  irritation.  Hankin 
arrived  at  the  view  that  the  bacillus  first  produces  a  ferment  and 
then  elaborates  albumoses.  In  tetanus  the  bacillus  produces  a 
secretion  of  non-proteid  toxin  which  causes  the  convulsions.  The 
albumoses  present  in  this  disease  are  probably  due  to  the  secretory 
toxin.  Ehrlich  has  isolated  a  spasm-producing  toxin  (tetanospasmin), 
and  a  crude  poison  capable  of  destroying  red  blood  cells  (tetanolysin). 
The  nature  of  the  tetanus  toxin  is  not  determined,  but  it  is  known 
that  it  is  a  most  powerful  poison,  probably  less  than  ^^th  of  a  grain 
being  poisonous  to  man.  In  diphtheria,  too,  we  have  a  secretory 
poison  in  the  membrane  and  in  the  tissues,  and  an  albumose  which 
is  possibly  the  result  of  the  secretion.  But  the  true  chemical  nature 
of  the  diphtheria  toxin  is  also  still  unknown.  In  typhoid  fever 
intra-cellular  bacillary  poisons  exist,  and  a  toxalbumin  has  been 
obtained  which  has  pathogenic  effects  of  an  indefinite  character. 
The  toxins  of  the  typhoid  bacillus  appear  to  have  little  digestive 
effect. 

Summary  of  Toxic  Effects. — The  action  of  bacteria  as  disease 
producers  depends  (1)  upon  the  effects  of  the  presence  of  the  bacteria 
themselves,  and  (2)  upon  their  power  of  forming,  directly  or  indirectly, 
certain  chemical  organic  products  known  as  toxins.  The  effects  of 
the  bacteria,  though  very  diverse,  may  be  classified  generally  as  of  a 
necrotic  or  a  separative  character,  leading  to  increased  functional 
activity  at  first  (such  as  phagocytosis),  and  subsequently  to  in- 
creased formative  activity  (such  as  cell  growth  and  subdivision). 
In  most  diseases  the  lesion  has  a  special  site  (as  in  typhoid  fever), 
and  the  body  generally  is  only  affected  indirectly.  This  locali- 
sation may  be  due  to  specific  action,  or  to  point  of  entrance  of 
the  bacillus  (as  in  malignant  pustule).  Secondarily  to  tissue 


410     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

changes,  the  body  metabolism  is  affected  owing  to  the  distribution 
of  toxins,  and  it  is  to  this  cause  that  the  chief  symptoms  of  disease 
are  due. 

The  Question  of  Immunity 

However  the  details  of  the  modus  operandi  of  the  formation  of 
toxins  are  finally  settled,  we  know  that  there  comes  a  time  when 
the  disease  symptoms  vanish,  the  disease  declines,  and  the  patient 
recovers.  In  past  times  this  was  explained  by  saying  that  the 
disease  had  exhausted  itself,  having  gone  "through"  the  body.  In 
a  sense  that  idea  is  probably  true ;  but  recently  a  number  of 
investigators  have  applied  themselves  to  this  problem,  and  with 
some  promising  results.  And  it  is  now  known  that,  as  a  result 
of  the  action  of  the  toxins  in  the  body  tissues,  powers  of 
resistance  are  stimulated  or  conferred  in  or  upon  the  body  cells 
affected.  What  has  been  found  to  be  true  of  lower  animals  by 
experimentation  is  now  known  to  be  true  of  the  human  body.  It 
has,  therefore,  become  possible  to  inoculate  resistant  blood  serum 
into  toxic  blood  with  the  result  of  opposing  the  toxins,  and  bringing 
about  a  condition  of  resistance,  and  ultimately,  recovery.  Or,  in 
other  words,  one  of  the  means  of  defence  against  the  invasion  of 
such  organisms  which  is  possessed  by  the  animal  body  is  the 
capacity  to  manufacture,  and  set  free  in  the  blood  stream,  sub- 
stances which  combine  with  the  toxins  and  so  render  them  inert. 
By  habituating  a  large  animal,  such  as  the  horse,  to  the  action  of 
toxin  in  increasing  quantities,  cells  or  fluids  of  its  body  can  be 
thereby  so  stimulated  to  produce  and  throw  into  the  blood  stream 
antitoxins  in  excessive  quantity,  that  the  serum  of  the  animals  may 
contain  sufficient  excess  for  its  useful  employment  as  a  remedy  for 
the  disease  in  man  or  animals.  From  such  results  it  is  but  a  step 
to  protective  inoculation. 

Various  protective  inoculations  against  anthrax,  for  instance, 
were  practised  as  early  as  1881,  and  the  protected  animals  remained 
healthy.  In  1887  Wooldridge  succeeded  in  protecting  rabbits  from 
anthrax  by  a  new  method,  by  which  he  showed  that  the  growth  of 
the  anthrax  bacillus  in  special  culture  fluids  gave  rise  to  a  substance 
which,  when  inoculated,  conferred  immunity.  In  1889  and  1890 
Hankin  and  Ogata  worked  at  the  subject,  and  announced  the 
discovery  in  the  blood  of  animals  which  had  died  of  anthrax  of 
substances  which  appeared  to  have  an  antagonistic  and  neutralising 
effect  upon  the  toxins  of  anthrax  and  upon  the  anthrax  bacilli 
themselves.  These  substances,  they  afterwards  found,  were  products 
of  the  anthrax  bacillus.  Behring  and  Kitasato  arrived  at  much  the 
same  results  in  tetanus  and  diphtheria.  In  1890  they  showed  that 
the  blood  serum  of  an  animal  which  had  been  immunised  against 


PRODUCTION  OF  ANTITOXIN  411 

tetanus  was  capable,  when  injected  into  other  animals,  of  protecting 
them  not  only  against  poisoning  with  tetanus  toxin  but  also  against 
infection  with  living  tetanus  bacilli.  They  also  proved  that,  under 
certain  conditions,  a  curative  action  could  be  demonstrated  in  animals 
which  already  presented  symptoms  of  tetanus  infection.  Similar, 
though  less  striking,  results  were  described  in  the  case  of  diphtheria. 
The  next  step  was  to  isolate  these  substances,  and  separating 
them  from  the  blood,  investigate  still  further  their  constitution. 
A  number  of  workers  were  soon  occupied  at  this  task,  and 
Buchner,  Hankin,  the  Klemperers,  Eoux,  Sidney  Martin,  and 
others  have  added  to  our  knowledge  respecting  these  toxin- 
opposing  bodies  now  known  as  antitoxins.  Some  believed  these 
bodies  were  a  kind  of  ultratoxin — substances  of  which  an  early 
form  was  a  toxin ;  others  held  that,  as  the  toxins  were  products  of 
the  bacteria  invading  the  tissues,  the  antitoxins  were  of  the  nature 
of  ferments  produced  by  the  resisting  tissues.  A  third  view  is  that 
possibly  antitoxins  may  be  the  result  of  an  increased  formation  of 
molecules  normally  present  in  the  tissues.  Finally,  antitoxins  came 
to  be  looked  upon  as  protective  substances  produced  in  the  body  cells 
as  a  result  of  toxic  action,  and  held  in  solution  in  the  blood,  and 
there  and  elsewhere  exerting  their  influence  in  opposition  to  the 
toxins.  These  antitoxic  bodies  gradually  increase  in  the  blood  and 
tissues,  and  their  action  falls  into  two  groups :  (a)  antitoxic,  which 
counteract  the  effects  of  the  poison  itself;  and  (b)  antimicrobic, 
which  counteract  the  effects  of  the  bacillus  itself.  "In  one  and 
the  same  animal  the  blood  may  contain  a  substance  or  substances 
which  are  both  antitoxic  and  antimicrobic,  such,  for  example,  as 
occurs  in  the  process  of  the  formation  of  the  diphtheria  and 
tetanus  antitoxic  serums "  (Sidney  Martin).  Antitoxin  must, 
therefore,  be  looked  upon  as  a  normal  constituent  of  the  living 
cells  which  is  produced  in  increased  quantity.  Of  the  chemical 
nature  of  toxins  and  antitoxins,  very  little  is  known.  Martin 
and  Cherry  have  come  to  the  conclusion  that  toxins  are  prob- 
ably of  the  nature  of  albumoses,  and  antitoxins  probably  have 
a  molecule  of  greater  size,  and  may  be  allied  to  the  globulins. 
Antitoxin  has  been  shown  to  appear  in  the  various  secretions 
of  the  body  as  well  as  in  the  blood,  though  in  a  less  concentrated 
state. 

The  relation  of  the  antitoxin  to  the  toxin,  and  its  mode  of 
antagonism,  is  probably  one  analogous  to  chemical  union.  The  two 
bodies  unite  to  form  an  inert  compound  possessing  no  toxic  or 
pathogenic  effect.  It  is  found  that  a  definite  period  of  time  elapses 
before  the  effect  of  the  toxin  is  neutralised,  and  that  it  takes  place 
more  rapidly  in  strong  solutions  than  in  weak,  and  in  warm 
temperature  than  in  cold,  which  all  goes  to  confirm  the  view  that 


412     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

such  union  is  the  mode  of  antagonism.*  The  progress  of  disease 
is,  therefore,  a  struggle  between  the  toxins  and  the  antitoxins: 
when  the  toxins  are  in  the  ascendency  we  have  an  increase  of 
the  disease  *  when  the  antitoxins  are  in  the  ascendency  we  have  a 
diminution  of  disease.  If  the  toxins  triumph,  the  result  is  death ; 
if  the  antitoxins  and  resistance  of  the  tissues  triumph,  the  result 
is  recovery. 

Different  Kinds  of  Immunity. — We  have  gathered,  then,  that 
whenever  bacteria,  introduced  into  the  blood  and  tissues,  fail  to 
multiply  or  produce  infection  (as  in  saprophytic  bacteria,  or  in 
immunity  of  a  particular  animal  from  a  specific  microbe),  this 
inability  to  perform  their  role  is  brought  about  by  some  property 
in  the  living  blood  serum  which  opposes  their  life  and  action ; 
and  further  we  have  seen  that  this  protective  property  is  ex- 
haustible according  to  the  number  of  bacteria,  and  differs  with 
various  species  of  bacteria,  and  in  different  animals.  Buchner 
designated  the~se  protective  bodies,  held  in  solution  in  the  blood, 
alexines,  and  regarded  them  as  belonging  to  the  albuminous  bodies 
of  the  lymph  and  plasma.  Alexines  are  naturally  produced  anti- 
toxins; ordinary  antitoxins  are  acquired  alexines.  Hence  we  have 
the  well-known  terms  "  natural "  and  "  acquired  "  immunity.  Of  the 
former  we  have  already  spoken.  The  latter,  acquired  immunity,  is 
a  protection  not  belonging  to  the  tissues  of  individuals  naturally  and 
as  part  of  their  constitution,  but  it  is  acquired  during  their  lives  as 
a  further  protection  of  the  tissues.  This  may  happen  in  one,  or 
both,  of  two  ways.  Either  it  may  be  an  involuntary  acquired 
immunity,  or  a  voluntary  acquired  immunity,  a  natural  attack  of 
disease,  or  an  artificial  attack  due  to  inoculation.  Small-pox,  typhoid 
fever,  even  scarlet  fever,  are  diseases  which  rarely  attack  the  same 
individual  twice.  That  is  because  each  of  these  diseases  leaves 
behind  it,  so  to  speak,  its  antitoxic  influence.  Hence  the  individual 
has  involuntarily  acquired  immunity  against  these  diseases.  An 
example  of  voluntary  acquired  immunity  is  also  at  hand  in  the  old 
method  of  preventive  inoculation  for  small-pox,  or  variolation.  This 
was  clearly  an  inoculation  setting  up  an  artificial  and  mild  attack 
of  small-pox,  by  which  the  antitoxins  of  that  disease  were  produced, 
and  protected  the  individual  against  further  infection  of  small-pox ; 
that  is  to  say,  it  was  a  voluntary  acquired  immunity.  This  form  of 
artificial  production  of  protection  is  artificial  immunity.  It  may  be 

*  Ehrlich  has  shown  that  the  antitoxic  power  of  these  anti-bodies  varies  widely, 
and  is  not  uniform.  Moreover,  antitoxins  are  specific  in  their  action.  He  suggests 
that  the  ultimate  toxin  molecule  contains  two  unsatisfied  affinities,  one  of  which  can 
•combine  with  antitoxin  (haptophorous),  and  the  others  having  a  toxic  action 
(toxophorous).  These  groups  under  certain  conditions  can  lose  none  of  their  combin- 
ing power,  the  toxophorous  being  more  readily  weakened  than  the  haptophorous. 
The  weakened  toxins  are  termed  toxoids  or  toxones. 


THEORIES  OF  IMMUNITY  413 

convenient  to  marshal  together  these  various  terms  in  a  table  as 
follows  :  — 

Immunity  in  manj  =  a  c°ndition  of  protection  or  insusceptibility  to  certain 

1.  Natural  immunity  =  constitutional  protection  produced  by  alexines. 

Acquired  naturally  (involuntary)  produced  by  anti- 

toxins formed*  by  an  attack  of  the  disease. 
Acquired  artificially  (voluntary)  = 
(a)  Active  immunity,  produced  by  direct  inocula- 
tion of  the  weakened  bacteria  or  weakened 
toxins  of  the  disease,   e.g.   vaccination,  or 
Pasteur's  treatment  of  rabies,  or  Haffkine's 

.     ,  .  .,  inoculation  for  cholera. 

Acquired  immunity^ 


(ft)  pa^  immunity>  produced  by  inoculation, 
not  of  the  disease  or  of  its  toxins,  but  of  the 
antitoxins  produced  in  the  body  of  an  animal 
suffering  from  the  specific  disease.  These 
antitoxins  combine  in  some  way  with  the 
toxins,  and  so  avert  their  harmful  effects. 
An  example  of  passive  immunity  occurs  in 
diphtheria  antitoxin. 

Theories  of  Immunity 

We  may  now  consider  shortly  how  these  new  facts  were  received, 
and  what  theories  of  explanation  were  put  forward  to  explain  con- 
tinued insusceptibility  to  disease.  It  had,  of  course,  been  known  for 
a  long  time  that  one  attack  of  small-pox,  for  example,  in  some 
degree  protected  the  individual  from  a  subsequent  attack  of  the 
same  disease.  To  that  experience  it  was  now  necessary  to  add  a 
large  mass  of  experimental  evidence  with  regard  to  toxins  and 
antitoxins.  The  chief  theories  of  immunity  which  have  been  pro- 
pounded are  as  follows  :  — 

1.  The  Exhaustion  Theory.  —  The  supporters  of  this  view  argued 
,  that  bacteria  of  disease  circulating  in  the  body  exhausted  the  body  of 

the  supply  of  some  pabulum  or  condition  necessary  for  the  growth 
and  development  of  their  own  species  (Pasteur). 

2.  The  Retention  Theory.  —  This  theory,  on  the  contrary,  was  based 
upon  the  view  that  there  were  certain  products  of  micro-organisms 
of  disease  retained  in  the  body  after  an  attack  which  acted  antagon- 
istically to  the  further  growth  in  the  body  of  that  same  species,  as 
occurs  in  a  test-tube  culture. 

3.  The  Acquired   Tolerance    Theory.  —  Some  have   advanced   the 
theory  that,  after  a  certain  time,  the  human  tissues  acquired  such 
a  degree  of  tolerance  to  the  specific  bacteria  or  their  specific  products, 
that  no  result  followed  their  action  in  the  body.     The  tissues  became 
acclimatised  to  the  disease. 

4.  The  Phagocyte  Theory.  —  This  theory,  which  gained  so  many 
adherents   when   first  promulgated   by  Metchnikoff,    attributes    to 


414     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

certain  cells  in  the  tissues  the  powers  of  "  scavenging,"  overtaking 
germs  of  disease,  and  absorbing  them  into  their  own  protoplasm. 
This,  indeed,  may  be  actually  witnessed,  and  had  been  observed  before 
the  time  of  Metchnikoff.  But  he  it  was  who  applied  the  observation 
to  the  destruction  of  pathogenic  organisms.  He  came  to  the  con- 
clusion that  the  successful  resistance  which  an  animal  offered  to 
bacteria  depended  upon  the  activity  of  these  scavenging  cells,  or 
phagocytes.  These  cells  are  derived  from  various  cellular  elements 
normally  present  in  the  body :  leucocytes,  endothelial  cells,  connective 
tissue  corpuscles,  and  any  and  all  cells  in  the  body  which  possess 
the  power  of  ingesting  bacteria.  If  they  were  present  in  large 
numbers  and  active,  it  was  argued,  the  animal  was  insusceptible  to 
certain  diseases;  if  they  were  few  and  inactive,  the  animal  was 
susceptible.  It  appears  that  the  bacteria  or  other  foreign  bodies  in 
the  blood  which  are  attacked  by  the  phagocyte  become  assimilated 
until  they  are  a  part  of  the  phagocyte  itself.  Metchnikoff  explained 
how  the  phagocyte  is  able  to  encounter  bacteria  when  both  are 
circulating  through  the  blood.  It  is  guided,  he  holds,  in  this  attack 
on  the  organisms  by  the  power  of  chemiotaxis.  The  bacteria  elaborate 
a  chemical  substance  which  attracts  the  phagocyte,  and  this  is 
termed  "  positive  chemiotaxis."  But  it  may  occur  that  the  chemical 
substance  produced  by  the  bacteria  may  have  an  opposite,  or  repellent, 
effect  upon  the  leucocytes,  in  which  case  we  have  "  negative  chemio- 
taxis." Metchnikoff  distinguishes  two  chief  varieties  of  phagocytes 
which  become  active  in  disease :  (a)  the  microphages,  which  are  the 
polynuclear  leucocytes  of  the  blood,  and  (&)  the  macrophages,  which 
include  the  larger  hyaline  leucocytes,  connective  tissue  cells,  etc.  It 
is  now  known  that  blood  serum,  from  which  all  leucocytes  (phagocytes) 
have  been  removed,  possesses  immunising  effects  as  before,  it  is 
therefore  clear  that  such  effect  is  a  property  of  the  serum  per  se,  and 
not  wholly  or  only  due  to  the  scavenging  power  of  certain  cells  in  it. 
Metchnikoff  explains  this  fact  by  stating  that  the  phagocytes  possess 
digestive  ferments  (cytases)  which  may  be  set  free  in  the  blood 
serum,  giving  it  its  bactericidal  properties.  Metchnikoff  admits  that 
antitoxin  and  toxin  form  a  neutral  compound,  but  holds  also  that 
acquired  resistance  of  body  cells  is  of  importance  in  toxin  immunity. 
5.  Ehrlich's  Side  Chain  Theory. — Ehrlich  looks  upon  a  molecule 
of  protoplasm  as  composed  of  a  central  atom  cell  with  a  large 
number  of  side  chains  of  atom  groups.  The  central  cell  is  the 
mother  cell,  the  side  chains  are  receptors,  that  is,  cells  having  com- 
bining affinity  with  food  stuffs  by  which  nutriment  is  brought  to 
the  mother  cell.  These  receptors  are  of  two  kinds,  those  having 
power  of  combining  with  molecules  of  simple  constitution,  and  those 
having  power  of  breaking  up  compound  bodies  by  ferment  action  for 
the  purposes  of  assimilation.  Now  if  toxins  be  introduced  into  the 


VACCINATION  415 

system  they  are  fixed  to  the  receptors  by  their  haptophorous 
elements,  and  their  toxophorous  elements  are  therefore  free,  and  if 
in  sufficient  numbers  or  amount  produce  the  toxic  changes.  If  the 
dose  of  toxin  molecules  is  small,  the  mother  cell  is  able  to  throw  off 
the  receptor  plus  the  toxin  (E  +  T),  which  thereby  becomes  free  in 
the  blood.  The  central  atom  group,  however,  is  able  to  produce  new 
receptors,  which  in  their  turn  come  to  be  free  in  the  blood.  As  a 
result  of  repeated  loss,  the  regeneration  of  receptors  becomes  an  over- 
regeneration,  and  the  excess  of  unfixed  receptors  become  free  in  the 
blood,  constituting  antitoxin  molecules.  When  forming  part  of  the 
mother  cell  the  receptors  anchor  the  toxin  which  is  thus  able  to  set 
up  toxic  effects  in  the  body  cells  and  tissues,  but  when  the  receptors 
are  free  in  the  blood  (E  +  T),  we  have  an  inert  compound,  and 
therefore  no  toxic  effect.  This  ingenious  theory  of  Ehrlich  explains 
the  facts  of  antitoxic  effect  better  than  any  other,  and  though  not 
established,  and  still  requiring  much  more  elucidation,  is  the  theory 
which  mostly  holds  the  field  at  the  present  time. 

The  Application  of  the  Principles  of  Immunity 

We  propose  now  to  consider  in  some  detail  four  illustrations  of 
the  application  of  the  facts  concerning  immunity  to  the  prevention 
or  treatment  of  disease,  viz.,  vaccination,  Pasteur's  treatment  of 
rabies,  antityphoid  and  antiplague  inoculation,  and  antitoxin  inocu- 
lation for  diphtheria.  The  vaccination  in  small-pox  is  an  inoculation 
of  the  virus  of  an  attenuated  form  of  the  disease  ;  the  rabies  inocula- 
tion is  a  transmission  of  the  vital  products  of  the  attenuated  disease ; 
the  typhoid  and  plague  inoculations  are  of  pure  cultures  of  living 
virus  from  outside  the  body ;  and  the  diphtheria  inoculation  is  the 
introduction  of  antitoxins  (passive  immunity).* 

Vaccination  for  Small-pox 

In  1717,  Lady  Mary  Wortley  Montaguf  described  the  inoculation 
of  small-pox  as  she  had  seen  it  practised  in  Constantinople.  So 
greatly  was  she  impressed  with  the  efficacy  of  this  process,  that  she 
had  her  own  son  inoculated  there,  and  in  1721,  Mr  Maitland,  a 
surgeon,  inoculated  her  daughter  in  London.  This  was  the  first  time 
inoculation  was  openly  practised  in  England.  J  For  one  hundred  and 
twenty  years  small-pox  inoculation  (or  variolation,  as  it  is  more 

*  See  also  Serums,  Vaccines,  and  Toxines,  W.  C.  Bosanquet,  1904. 

t  The  friend  of  Addison  and  Pope,  who  married  Mr  Edward  Wortley  Montagu 
in  1712,  and  on  his  appointment  to  the  ambassadorship  of  the  Porte  in  1716  went 
with  him  to  Constantinople.  They  remained  abroad  for  two  years,  during  which 
time  Lady  Wortley  Montagu  wrote  her  well-known  Letters  to  her  sister  the  Countess 
of  Mar,  Pope,  and  others. 

£  Crookshank,  History  and  Pathology  of  Vaccination. 


416     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

correctly  termed)  was  practised  in  this  country,  until  by  Act  of 
Parliament  in  1840  it  was  prohibited.  There  were  different  methods 
of  performing  variolation,  but  the  most  approved  was  similar  to  the 
modern  system  of  arm-to-arm  vaccination,  the  arm  being  inoculated, 
by  a  lancet  in  one  or  more  places,  with  small-pox  lymph  instead  of, 
as  now,  with  vaccine  lymph.  As  a  rule,  only  local  results  or  a 
mild  attack  of  small-pox  followed,  which  prevented  an  attack  of 
natural  small-pox.  But  its  disadvantage  is  apparent :  it  was  in  fact 
inoculating  small-pox,  and  it  was  a  means  of  breeding  small-pox, 
for  the  inoculated  cases  were  liable  to  create  fresh  centres  of  infection. 

In  1796,  Edward  Jenner,  who  was  a  country  practitioner  in 
Gloucestershire,  observed  that  those  persons  affected  with  cow-pox, 
contracted  in  the  discharge  of  their  duty  as  milkers,  did  not  contract 
small-pox,  even  when  placed  in  risk  of  infection.  Hence  he  inferred 
that  inoculation  of  this  mild  and  non-infectious  disease  would  be 
protective  against  small-pox,  and  would  be  preferable  to  the  process 
of  variolation  then  so  widely  adopted  in  England.  Jenner  therefore 
suggested  the  substitution  of  cow-pox  lymph  (vaccine)  in  place  of 
small-pox  lymph,  as  used  in  ordinary  variolation. 

It  should  not  be  forgotten  that  variolation  was  thus  the  first 
work  done  in  this  country  in  producing  artificial  immunity,  and 
was  followed  by  vaccination,  which  was  only  partly  understood. 
Even  to-day  there  is  probably  much  to  learn  respecting  it.  Vaccina- 
tion may  be  defined  as  active  immunisation  by  means  of  a  weakened 
form  of  the  specific  virus  causing  the  disease.  The  nature  of  the 
specific  virus  of  both  small-pox  and  cow-pox  awaits  discovery. 
Burdon  Sanderson,  Crookshank,  Klein,  Copeman,  and  others  have 
demonstrated  bacteria  in  cow-pox  or  vaccine  lymph,  and  in  1898 
Copeman  announced  that  he  had  isolated  a  specific  bacillus  and 
grown  it  upon  artificial  media.*  Numerous  statements  have  been 
made  to  the  effect  that  a  specific  bacillus  has  been  found  in  small-pox 
also.  But  neither  in  small-pox  nor  cow-pox  is  the  nature  of  the 
contagium  really  known.f 

These  facts,  however,  did  not  remove  the  suspicion  which  had 
hitherto  rested  upon  vaccine  lymph  as  a  vehicle  for  bacteria  of  other 
diseases  which  by  its  inoculation  might  thus  be  contracted.  A  few 
remarks  are  therefore  called  for  at  this  juncture  upon  the  work  of 
Copeman  and  Blaxall,  in  respect  to  what  is  known  as  glycerinatcd 
calf  lymph.  Evidence  has  been  forthcoming  to  substantiate  in  some 
measure  the  distrust  which  many  persons  have  from  time  to  time 

*  An  exhaustive  account  of  vaccine  may  be  found  in  the  Milroy  lectures,  delivered 
in  1898  at  the  Royal  College  of  Physicians  by  S.  Monckton  Copeman,  M.D.,  Brit. 
Med.  Jour.,  1898,  vol.  i.,  pp.  1185,  1245,  1312  ;  see  also  paper  on  the  "  Bacteriology 
of  Vaccinia  and  Variola,"  Brit.  Med.  Jour.,  1902,  vol.  ii.,  pp.  52-67. 

|  Crookshank,  Bacteriology  and  Infective  Diseases ;  Virchow,  The  Huxley  Lecture, 
1  S9S. 


EFFECT  OF  VACCINATION  417 

felt  in  the  vaccine  commonly  used  in  vaccination,  hence  the  new 
form  as  above  designated.  This  retains  the  toxic  qualities  required 
for  immunity,  but  is  so  produced  that  it  possesses  in  addition  three 
very  important  advantages  :  namely,  it  is  entirely  free  from  extran- 
eous organisms,  it  is  available  for  a  large  number  of  vaccinations,  and 
it  retains  full  activity  for  eight  months.  It  is  prepared  as  follows : 
— A  calf,  aged  three  to  six  months,  is  kept  in  quarantine  for  a  week. 
If  then  found  upon  examination  to  be  quite  healthy,  it  is  removed 
to  the  vaccinating  station,  and  the  lower  part  of  its  abdomen  anti- 
septically  cleaned.  The  animal  is  now  vaccinated  upon  this  sterilised 
area  with  glycerinated  calf  lymph.  After  five  days  the  part  is  again 
thoroughly  washed,  and  the  contents  of  the  vesicles,  which  have  of 
course  appeared  in  the  interval,  are  removed  with  a  sterilised  sharp 
spoon,  and  transferred  to  a  sterilised  bottle.  This  is  now  removed  to 
the  laboratory,  and  the  exact  weight  of  the  material  ascertained.  A 
calf  thus  vaccinated  will  yield  from  18  to  24  grams  of  vaccine 
material.  This  is  now  thoroughly  triturated  and  mixed  with  six 
times  its  weight  of  a  sterilised  solution  of  50  per  cent,  chemically 
pure  glycerine  in  distilled  water.  The  resulting  emulsion  is  asepti- 
cally  stored  in  sealed  tubes  in  a  cool  place.  At  intervals  during  four 
weeks  it  is  carefully  examined  bacteriologically  until  by  agar  plates 
it  is  demonstrably  free  from  extraneous  organisms,  when  it  is  ready 
for  distribution. 

The  Effect  of  Vaccination. — The  Royal  Commission  on  Vaccination,  1896, 
concluded  (p.  90)  that  the  protection  vaccination  affords  against  small-pox  may  be 
stated  as  follows  : — 

"  (1)  That  it  diminishes  the  liability  to  be  attacked  by  the  disease.  (2)  That  it 
modifies  the  character  of  the  disease  and  renders  it  less  fatal  and  of  a  less  severe 
type.  (3)  That  the  protection  it  affords  against  attacks  of  the  disease  is  greatest 
during  the  years  immediately  succeeding  the  operation  of  vaccination.  It  is 
impossible  to  fix  with  precision  the  length  of  this  period  of  highest  protection. 
Though  not  in  all  cases  the  same,  if  a  period  is  to  be  fixed,  it  might,  we  think,  fairly 
be  said  to  cover  in  general  a  period  of  nine  or  ten  years.  (4)  That  after  the  lapse  of 
the  period  of  highest  protective  potency,  the  efficacy  of  vaccination  to  protect  against 
attack  rapidly  diminishes,  but  that  it  is  still  considerable  in  the  next  quinquennium, 
and  possibly  never  altogether  ceases.  (5)  That  its  power  to  modify  the  character  of 
the  disease  is  also  greatest  in  the  period  in  which  its  power  to  protect  from  attack  is 
greatest,  but  that  its  power  thus  to  modify  the  disease  does  not  diminish  as  rapidly 
as  its  protective  influence  against  attacks,  and  its  efficacy  during  the  later  periods  of 
life  to  modify  the  disease  it  still  very  considerable.  (6)  That  revaccination  restores 
the  protection  which  lapse  of  time  has  diminished,  but  the  evidence  shows  that  this 
protection  again  diminishes,  and  that  to  ensure  the  highest  degree  of  protection  which 
vaccination  can  give  the  operation  should  be  at  intervals  repeated.  (7)  That  the 
beneficial  effects  of  vaccination  are  most  experienced  by  those  in  whose  case  it  has 
been  most  thorough.  We  think  it  may  be  fairly  concluded  that  where  the  vaccine 
matter  is  inserted  in  three  or  four  places  it  is  more  effectual  than  when  introduced  into 
one  or  two  places  only,  and  that  if  the  vaccination  marks  are  of  an  area  of  half  a 
square  inch  they  indicate  a  better  state  of  protection  than  if  their  area  be  at  all  con- 
siderably below  this." 

These  findings  are  well  illustrated  in  the  returns  of  the  London  Epidemic  of 
Small-pox  which  occurred  in  1901-2.  These  returns  are  the  most  recent  evidence  as 
to  the  protection  afforded  by  vaccination.  They  are  as  follows  : — 

2  D 


418     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 


Vaccinated. 

Unknown. 

Unvaccinated. 

Total  cases  and 

deaths. 

Ages. 

i 

i 

f! 

i 

1 

Mortality, 
per  cent. 

I 

1 

Mortality, 
per  cent. 

i 

I 

f  ! 

O  <D 

Years. 

Under  1     . 

187 

130 

69-52 

187 

130 

69-52 

1  to  5 

18 

7 

1 

14-28 

524 

209 

39-88 

549 

210 

38-25 

5  to  10      . 

116 

2 

1-72 

26 

5 

19-23 

563 

103 

18-29 

705 

110 

15-60 

10  to  15      . 

334 

4 

1-19 

29 

5 

17-24 

386 

88 

22-79 

749 

97 

12-95 

15to20      . 

829 

19 

2-29 

44 

10 

22-73 

233 

62 

26-61 

1106 

91 

8-22 

Total  under  20 

1297 

25 

1-93 

106 

21 

19-81 

1893 

592 

31;27 

3296 

638 

19-35 

20  to  25      . 

1274 

60 

4-71 

49 

16 

32-65 

149 

47 

31-54 

1472 

123 

8-35 

25  to  30      . 

1243 

87 

7-00 

49 

24 

48-98 

94 

38 

40-43 

1386 

149 

10-75 

30  to  35      . 

997 

111 

11-13 

45 

21 

46-67 

52 

22 

42-31 

1094 

154 

14-07 

35  to  40      . 

758 

137 

18-07 

32 

15 

46-87 

38 

19 

50-00 

828 

171 

20-65 

40  to  50      . 

893 

188 

21-05 

62 

33 

53-23 

31 

24 

77-42 

986 

245 

24-84 

50  to  60      . 

320 

61 

19-06 

52 

23 

44-23 

13 

8 

61-54 

385 

92 

23-89 

60  to  70      . 

126 

30 

23-8 

25 

8 

32-00 

5 

156 

38 

24-39 

70  to  80      . 

31 

5 

16-13 

15 

9 

60-00 

1 

1 

100-00 

47 

15 

31-91 

Over  80      . 

6 

1 

16-67 

1 

1 

100-00 

1 

1 

100-00 

8 

3 

37-50 

Total  between! 

20  and  80  J 

5648 

680 

12-04 

330 

150 

45-45 

384 

160 

41-66 

6362 

990 

15-56 

Grand  Total  . 

6945 

705 

10-15 

436 

171 

39-22 

2277 

752 

33-03 

9658 

1628 

16-85 

From  these  figures  it  will  be  seen  :— 

(a)  That  under  five  years  of  age  18  cases  of  small-pox  occurred  in  children  who 
had  been  vaccinated,  whilst  711  cases  occurred  in  children  who  had  not  been 
vaccinated.  (6)  That  under  ten  years  of  age  the  cases  of  small-pox  in  vaccinated 
persons  had  a  mortality  percentage  of  1*72,  and  the  unvaccinated  cases  had  a 
mortality  percentage  of  over  42  per  cent,  (c)  That  under  twenty  years  of  age  the 
cases  of  small-pox  occurring  in  vaccinated  persons  had  a  mortality  percentage  of  1  -93, 
and  the  unvaccinated  cases  had  a  mortality  percentage  of  31  -27.  (d)  That  over 
twenty  years  of  age  the  cases  of  small-pox  occurring  in  vaccinated  persons  numbered 
5648,  and  there  were  680  deaths,  giving  a  mortality  percentage  of  12*04 ;  whereas  the 
cases  occurring  in  unvaccinated  persons  were  384,  160  of  whom  died,  giving  a 
mortality  percentageof  41  -66.  The  larger  number  of  cases  of  small-pox  in  vaccinated 
persons  is,  of  course,  due  to  the  fact  that  by  far  the  larger  proportion  of  the  popula- 
tion at  that  age-period  have,  at  some  time  or  other  in  their  lives,  been  vaccinated. 
Three  broad  facts  stand  out  with  clearness : — (1)  That  small-pox  among  the 
vaccinated  is  nowadays  mainly  a  disease  of  adults,  because  children  are  protected 
by  primary  vaccination  and  adults  are  not  protected  by  re  vaccination.  (Ninety-two 
percent,  of  the  vaccinated  cases  were  over  fifteen  years  of  age.)  (2)  That  among 
the  unvaccinated,  small-pox  is  still,  in  great  measure,  a  disease  of  the  young  as  it  was  in 
prevaccination  days.  (Seventy-three  per  cent,  of  the  unvaccinated  cases  were  under 
fifteen  years  of  age.)  (3)  That  the  mortality  rate  among  the  vaccinated  is  at  all 


EFFECT  OF  VACCINATION 


419 


a>jes  much  less  than  among  the  unvaccinated,  and  that  this  difference  is  very  striking 
and  complete  in  children  because  of  their  recent  vaccination. 

Those  who  advocate  vaccination  and  revaccination  as  protective  in  a  greater  or 
lesser  degree  against  small-pox  do  so  upon  three  main  grounds.  In  the  first  place, 
they  claim  that,  other  things  being  equal,  persons  who  have  been  vaccinated  (especi- 
ally within  ten  years)  are  less  liable  to  attack  from  small-pox.  This  is  abundantly 
established  by  the  figures  quoted  above.  In  the  second  place,  they  claim  that 
persons  who  have  been  vaccinated,  and  yet,  on  account  of  their  greater  number  in 
the  population,  and,  therefore,  their  consequent  greater  probability  of  infection,  are 
attacked  by  small-pox,  do  not  die  so  readily  from  the  disease  as  those  who  have  not 
been  vaccinated.  This  claim  also  is  more  than  proved  in  the  returns  quoted  above. 
In  the  third  place,  they  claim  that  the  protection  afforded  by  vaccination  depends 
upon  the  efficiency  of  the  vaccination.  This  may  be  measured,  as  is  frequently  done, 
by  the  number  of  marks,  but  it  is  more  satisfactorily  measured  by  area  of  vaccination 
mark  (i.e.  area  of  cicatrix).  The  return  of  the  Metropolitan  Asylums  Board  respect- 
ing this  point  is  given  below,  and  a  study  of  it  will  amply  prove  the  claim  made. 


Admissions. 

Deaths. 

Mortality, 
per  cent. 

VACCINATED  CASES  — 

Area  of  Cicatrix  : 

Half  and  upwards  of  half 

square  inch 

5163 

379 

7'34 

Area  of  Cicatrix  : 

One-third,  but  less  than  half 

square  inch 

835 

131 

15-69 

Area  of  Cicatrix  : 
Less  than  one-third  square 

inch          .... 

860 

162 

16-87 

Area  of  Cicatrix  : 

Not  recorded      ;*-.-*       * 

87 

33 

37-93 

Totals  of  Vaccinated  Class 

6945 

705 

10-15 

UNKNOWN  AND  DOUBTFUL  CLASS  . 

436 

171 

39-22 

UNVACCINATED  CLASS   .        .    '    . 

2277 

752 

33-06 

Grand  Totals 

9658 

1628 

16-87 

Pasteur's  Treatment  of  Rabies 

Eabies  is  a  disease  affecting  dogs  (in  Western  Europe)*  and 
wolves  (in  Russia),  and  can  be  transmitted  to  other  animals  (chiefly 
mammals  and  especially  the  Carnivora)  and  man.  Infection  may 
be  conveyed  from  the  rabid  animal  by  biting  (which  is  the  most 
frequent  mode),  by  licking  raw  surfaces,  by  suckling,  and  possibly 
by  the  ingestion  by  animals  of  the  flesh  of  other  animals  which  have 
died  from  the  disease. 

*  In  the  decennium  1894-1903,  1555  dogs  in  Great  Britain  were  reported  as  suffer 
ing  from  rabies,  of  which  only  29  cases  occurred  during  the  last  five  years.  The 
marked  decline  in  recent  years  is  attributed  to  the  effects  of  the  muzzling  order  and 
stricter  inspection  of  ownerless  vagrant  dogs.  See  also  Year-Book  of  Department  of 
Agriculture,  U.S.A.,  1900. 


420     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

Although  rabies  was  mentioned  by  Aristotle,  and  has  been  studied 
by  a  large  number  of  workers  since,  the  contributions  of  Pasteur 
have  been  greater  than  all  the  other  additions  to  our  knowledge  of 
the  disease  put  together.  Professor  Eose  Bradford  has  pointed  out 
that  Pasteur's  discoveries  concerning  rabies  may  be  said  to  be  four 
in  number :  (a)  that  the  virus  was  not  only  in  the  saliva,  but  also  in 
the  central  and  peripheral  nervous  system,  yet  absent  from  the 
blood;  (b)  that  the  disease  was  most  readily  inoculated  in  the 
nervous  system;  (c)  that  by  suitable  means  the  virus  could  be 
attenuated ;  and  (d)  that  by  means  of  an  attenuated  virus  preventive 
and  even  curative  methods  might  be  adopted. 

The  disease  takes  two  chief  forms:  (1)  furious  rabies,  and  (2) 
paralytic  or  dumb  rabies.  The  former  is  more  common  in  dogs. 
The  animal  becomes  restless,  has  a  high-toned  bark,  and  snaps  at 
various  objects;  sometimes  it  exhibits  depraved  appetite.  Briefly, 
the  animal  passes  from  a  melancholy  to  a  maniacal  and  then  a 
paralytic  state,  ending  in  coma  and  death.  In  man,  the  incubation 
period  is  fortunately  a  very  long  one,  averaging  about  forty  days. 
Nervous  irritability  is  the  first  sign;  spasms  occur  in  the  respiratory 
and  masticatory  muscles,  and  the  termination  is  similar  to  rabies  in 
the  dog.  The  symptom  of  fear  of  water  is  a  herald  of  coming 
fatality. 

Although  a  number  of  the  workers  at  the  Pasteur  Institute  and 
elsewhere  have  addressed  themselves  to  the  detection  of  a  specific 
microbe,  none  has  as  yet  been  found,  although,  in  the  opinion 
of  Pasteur,  such  an  agent  may  be  suspected  as  the  cause. 

Pathologically,  rabies  and  tetanus  are  closely  allied  diseases,  and 
the  recent  remarkable  additions  to  our  knowledge  of  the  latter 
disease  only  make  the  similarity  more  evident.  There  are  in  rabies 
three  chief  sets  of  post-mortem  signs.  First,  and  by  far  the  most 
important,  are  the  changes  in  the  nervous  system.  Here  we  find 
patches  of  congestion  in  the  brain,  and  breaking  down  of  the  axis 
cylinders  of  the  nerves.  The  stomach,  in  the  second  place,  exhibits 
haemorrhagic  changes,  not  unlike  acute  arsenical  poisoning.  Thirdly, 
the  salivary  glands  show  a  degenerative  change  in  a  breaking  down 
of  their  secretory  cells.  Eoux  has  pointed  out  that  in  life  the 
saliva  of  a  mad  dog  becomes  virulent  three  days  before  the  appear- 
ance of  the  symptoms  of  disease.  The  poison  appears  to  be  present 
mainly  in  the  nervous  system  and  the  saliva ;  it  is  not  present  in 
the  blood. 

The  method  of  treatment  by  inoculation  was  introduced  by 
Pasteur.  Before  his  time  cauterisation  of  the  wound  was  the  only 
method  adopted.  But  if  more  than  half  an  hour  has  elapsed  since 
the  bite,  cauterisation  is  of  little  or  no  avail.  The  basis  of  Pasteur's 
treatment  was  the  difference  in  virulence  obtainable  in  spinal  cords 


PASTEUR'S  TREATMENT  FOR  RABIES 


421 


infected  with  rabies.  Pasteur  found  that  drying  the  cord  led  to  a 
lessening  of  its  virulence,  just  as  certain  other  conditions  increased 
its  virulence.  Next  he  established  the  fact  that  subcutaneous 
injection  of  a  weak  virus,  followed  up  with  doses  of  ever-increasingly 
virulent  cords,  immunised  dogs  against  infection  or  inoculation  of 
fully  virulent  material.  From  this  he  reasoned  that  if  he  could 
establish  a  standard  of .  weakened  virulence  he  would  have  at  hand 
the  necessary  "  vaccine  "  for  the  treatment  of  the  disease. 

Subsequent  research  and  skilled  technique  resulted  in  a  method 
of  securing  this  standard,  which  he  found  to  be  a  spinal  cord  dried 
for  fourteen  days.  The  exact  details  of  preparation  of  this  vaccine 
are  as  follows:  The  spinal  cords  of  two 
rabbits  dead  of  rabies  are  removed  from 
the  spinal  canal  in  their  entirety  by  means 
of  snipping  the  transverse  processes  of  the 
vertebrae.  Each  cord  is  divided  into  three 
more  or  less  equal  pieces,  and  each  piece, 
being  snared  by  a  thread  of  sterilised 
silk,  is  carefully  suspended  in  a  steril- 
ised glass  jar.  At  the  bottom  of  the  jar 
is  a  layer,  about  half  an  inch  deep,  of 
sterilised  calcium  chloride.  The  jars  are 
then  removed  to  a  dark  chamber,  where 
they  are  placed  at  a  temperature  of 
20-22°  C.  in  wooden  cases.  Here  they 
are  left  to  dry.  Above  each  case 
tube  of  broth,  to  which  has  been 
added  a  small  piece  of  the  corre- 
sponding cord,  in  order  to  test  for 
any  micro-organism  that  may  by 
chance  be  included.  In  case  of  the 
slightest  turbidity  in  the  broth,  the 
cord  is  rejected.  Fourteen  series  of 
cords  are  thus  suspended  on  four- 
teen consecutive  days.  The  first,  second,  and  third  are  found  to  be  of 
practically  equal  virulence,  but  from  the  third  to  the  fourteenth  the 
virulence  proportionally  decreases,  and  on  the  fifteenth  day  the  cord 
would  be  practically  innocuous  and  non-virulent.  When  treatment 
is  to  be  commenced,  obviously  the  weakest — that  is,  the  fourteenth 
day — cord  is  used  to  make  the  "vaccine,"  and  so  on  in  steadily 
increasing  doses  (as  regards  virulence)  up  to,  and  including,  a  third- 
day  cord.  The  fourteenth-day  cord  is  therefore  taken,  and  a  small 
piece  cut  off  and  extracted  in  10  c.c.  of  sterile  broth,  which  are 
placed  in  a  conical  glass  and  covered  with  two  layers  of  thick  filter- 
paper,  the  glass  with  its  covering  having  been  previously  sterilised 


is 


FIG.  3(5.— SUSPENDED  SPINAL  CORD. 
In  drying  jar  containing  Calcium  Chloride. 


422     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

by  dry  heat.  When  the  patient  bitten  by  the  rabid  animal  is 
prepared,  3  c.c.  of  this  broth  emulsion  of  spinal  cord  are  inoculated  by 
means  of  a  hypodermic  needle  (under  aseptic  precautions)  into  the 
flanks  or  abdominal  wall.  On  the  following  day  the  patient  returns 
for  an  inoculation  of  a  cord  of  the  thirteenth  day,  and  so  on  until  a 
rabid  cord  emulsion  of  the  first  three  days  has  been  inoculated.  As  a 
matter  of  practice,  the  dosage  depends  upon  the  three  recognised 
classes  of  bites,  viz.  (1)  bites  through  clothing  (least  severe);  (2) 
bites  on  the  bare  skin  of  the  hand ;  (3)  bites  upon  the  face  or  head, 
most  severe  owing  to  the  vascularity  of  these  parts.  An  example  of 
each,  which  the  writer  was  permitted  to  take  in  the  Pasteur  Institute, 
may  be  here  added  to  illustrate  the  usual  practice. 


Inoculation  Treatment  for  Persons  affected  with  Rabies. 


1.  For  those  Bitten  through 
Clothes. 

2.  For  those  Bitten  on 
Uncovered  Skin  of  Hands,  etc. 

3.  For  those  Bitten  on  Face 
or  Head. 

SJD 

tc 

&b 

Days  of 
Treatment. 

Doses  of 
Emulsion 
per  c.c. 

Dates  of 
Cord  Dryin 

Days  of 
Treatment. 

Doses  of 
Emulsion 
per  c.c. 

Dates  of 
Cord  Dryin 

Days  of 
Treatment. 

Doses  of 
Emulsion 
per  c.c. 

°'£> 
jl 

Si 

o 
O 

1  at  11  am. 

3 

14 

latll  a.ra. 

3 

14 

1  at  11a.m. 

3 

14 

1     „                   3 

13 

1     „ 

3 

13 

1     „       „ 

3 

13 

2     „ 

3 

12 

2     „ 

3 

12 

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12 

2     „ 

3 

11 

2     „ 

3 

11 

1     „       „ 

3 

11 

3     „ 

3 

10 

3     „ 

3 

10 

2  at  11a.m. 

3 

10 

3     „ 

3 

9 

3     „ 

3 

9 

2     „       „ 

3 

9 

4     „ 

3 

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4     „ 

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4     ,, 

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2     »»       »» 

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7 

5     11 

3 

6 

5     ,, 

3 

6 

3  at  11a.m. 

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5     „ 

3 

6 

3 

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6 

6     „ 

3 

5 

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3 

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4 

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7      , 

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3 

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3 

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2 

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16     „ 

3 

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17     „ 

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3 

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3 

5 

20 

3 

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21 

3 

3 

CHOLERA,  TYPHOID,  PLAGUE 


423 


Effect  of  Treatment.— It  may  be  well  to  add  the  returns  of  inoculation  made  at 
the  Pasteur  Institute,  Rue  Dutot,  Paris,  as  above  described,  and  the  mortality  rate 
resulting.  The  record  is  as  follows  : — 


Year. 

Number  of 
persons 
Inoculated. 

Number  of 
Deaths. 

Rate  of 

Mortality. 

1886                        • 

2671 

25 

0-94 

1887 

1770 

14 

0-79 

1888 

1622 

9 

0-55 

1889                . 

1830 

7 

0-38 

1890 

1540 

5 

0-32 

1891 

1559 

4 

0-25 

1892 

1790 

4 

0-22 

1893 

1648 

6 

0-36 

1894 

1387 

7 

0'50 

1895 

1520 

5 

0-33 

1896 

1308 

4 

0-30 

1897 

1521 

6 

0-39 

1898 

1465 

3 

0-20 

1899 

1614 

4 

0-25 

1900 

1420 

4 

0-35 

1901 

1321 

5 

0-38 

1902 

1105 

2 

0-18 

Of  the  1105  persons  under  treatment  in  1902,  9  were  English,  2  Spaniards,  2 
Russians,  and  one  each  Greek,  Dutch,  and  Swiss— making  16  foreigners,  1089  French. 
The  diminution  in  the  number  of  French  patients,  as  compared  with  several  preceding 
years,  is  explained  by  the  opening  of  anti-rabic  institutes  at  Lille,  Marseilles, 
Montpellier,  Lyons,  and  Bordeaux,  at  one  or  other  of  which  persons  residing  in  the 
neighbourhood  of  those  towns  have  been  sent  instead  of  going  to  Paris. 

Pasteur's  treatment  of  rabies  by  inoculation  of  emulsions  of  dried 
spinal  cord  is,  therefore,  a  "  vaccination  "  of  attenuated  virus,  result- 
ing in  antitoxin  formation,  to  the  further  protection  of  the  individual 
against  rabies. 

Inoculations  for  Cholera,  Typhoid,  and  Plague 

Anti-Cholera  Inoculation. — Inoculating  cholera  virus  against 
cholera  has  been  made  illegal,  like  variolation  was  in  1840.  But 
Haffkine  has  prepared  two  vaccines.  The  weak  one  is  made  from 
pure  cultures  of  Koch's  spirillum  of  Asiatic  cholera,  attenuated 
by  growth  to  several  generations  on  agar  or  broth  at  39°  C.,  or  by 
passing  a  current  of  sterile  air  over  the  surface  of  the  cultures. 
The  strong  one,  virus  exalte,  is  from  similar  culture  the  virulence  of 
which  has  been  increased  by  passage  through  guinea-pigs.  One 
cubic  centimetre  of  the  first  vaccine  is  injected  hypodermically  into 
the  flank,  and  the  second  vaccine  three  or  four  days  afterwards. 
The  immunisation  is  prophylactic,  not  remedial,  and  its  action  takes 
effect  five  or  six  days  after  the  second  vaccine  has  been  injected. 


424     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

Anti-Typhoid  Inoculation. — It  is  now  known  that  the  serum 
of  persons  who  have  recovered  from  typhoid  fever,  and  the  serum 
of  animals  artificially  immunised  against  virulent  typhoid  bacilli, 
protect  against  the  typhoid  bacillus.  Animals  have  now  been 
immunised  by  injections  of  the  toxins  of  the  typhoid  bacillus ;  and 
their  serum  aids  in  the  destruction  of  the  bacilli  which  produce  the 
toxins.  Acting  on  these  principles,  Wright  has  prepared  a  vaccine 
against  typhoid  fever.  A  virulent  twenty-four  hours  culture  is 
emulsified  in  bouillon,  and  killed  by  heating  for  five  minutes  at  60° 
C.  For  use,  one-twentieth  to  one-fourth  of  the  dead  culture  is 
injected  hypodermically,  usually  in  the  flank.*  The  effect  of  the 
inoculation  is  some  local  tenderness  and  swelling  with  enlargement 
of  adjacent  lymph  glands.  Within  ten  days  the  blood  of  the 
inoculated  person  begins  to  show  a  positive  Widal  reaction,  owing 
to  its  immunising  properties,  and  it  is  also  bactericidal  in  vitro. 

Haffkine's  Preventive  Inoculation  for  Plague.— In  plague 
the  same  plan  has  been  followed.  Luxurious  crops  of  Kitasato's 
plague  bacillus  are  grown  on  ordinary  broth  with  the  addition  to 
the  surface  of  a  film  of  oil  or  fat  ("  ghee  ").  Under  the  globules  of 
fat  flakes  of  plague  culture  grow  like  stalactites,  hanging  down  into 
the  clear  broth.  The  culture  is  kept  at  25°  C.  These  are,  every  few 
days,  shaken  to  the  bottom,  and  a  second  crop  grows  on  the  under- 
surface  of  the  fat.  In  the  course  of  six  weeks  a  number  of  such 
crops  are  obtained  and  shaken  down  into  the  fluid,  until  the  latter 
assumes  an  opaque  milky  appearance.  The  purity  of  this  culture 
is  controlled  by  transferring  with  a  sterile  pipette  a  small  quantity 
to  a  dry  agar  tube,  and  noting  the  appearance  of  the  growth  by 
reflected  light  through  the  thickness  of  the  agar.  The  culture  is 
now,  unlike  the  cholera  vaccine,  exposed  to  a  temperature  of  65°  C. 
for  one  hour  in  a  water-bath,  and  a  small  quantity  of  carbolic  acid  is 
added  ('5  per  cent.),  by  which  processes  the  bacilli  are  killed.  The 
dose  is  5  to  10  c.c.  This  preparation  has  the  advantage  of  being 
easily  prepared,  obtainable  in  large  quantities,  and  requires  no  animals 
in  its  preparation.  When  inoculated,  it  produces  local  pain  and 
swelling  at  the  site  of  inoculation,  and  general  reactive  symptoms  such 
as  fever.  From  a  careful  analysis  of  the  results  of  this  inoculation,  it 
is  shown  that  the  efficacy  of  the  prophylactic  depends  upon  the 
virulence  of  the  bacillus  culture  from  which  the  vaccine  is  prepared, 
and  upon  its  dose  and  ability  to  produce  a  well-marked  febrile 
reaction.  It  appears  to  be  more  effective  in  the  prevention  of  deaths 
than  of  attacks.f 

*  For  methods  employed  in  preparation  of  the  vaccine,  see  Brit.  Med.  Jour., 
1900,  vol.  i.,  p.  122  (Wright). 

t  Proc.  Roy.  Soc.,  1900  ;  Report  of  Medical  Officer  to  Local  Government  Board, 
1902,  pp.  357-94. 


DIPHTHERIA  ANTITOXIN  425 

The  Indian  Plague  Commission  concluded  that  (1)  inoculation 
sensibly  diminishes  the  incidence  of  plague  attacks  on  the  inoculated 
population,  but  the  protection  afforded  is  not  absolute ;  (2)  inocula- 
tion diminishes  the  death-rate  among  the  inoculated  population ;  (3) 
inoculation  does  not  appear  to  establish  protection  until  after  some 
clays ;  and  (4)  protection  is  conferred  for  a  considerable  number  of 
weeks  and  possibly  for  months.  Finally,  the  Commission  recom- 
mend that  under  the  safeguards  and  conditions  of  accurate 
standardisation  and  complete  sterilisation  of  the  vaccine,  and  the 
thorough  sterilisation  of  the  syringe  in  every  case,  inoculation  should 
be  encouraged  wherever  possible,  and  in  particular  among  disinfecting 
stuffs,  and  the  attendants  of  plague  hospitals. 

Antitoxin  Inoculation  for  Diphtheria 

We  may  now  consider  an  illustration  of  passive  immunity.  This, 
it  will  be  remembered,  may  be  defined  as-  a  protection  (against  a 
bacterial  disease)  produced  by  inoculation,  not  of  the  disease  itself, 
as  in  sinall-pox  inoculation,  nor  yet  of  its  weakened  toxins,  as  in 
rabies,  but  of  the  antitoxins  produced  in  the  body  of  an  animal 
suffering  from  that  particular  disease.  Examples  of  this  treatment 
are  increasing  every  year.  The  chief  examples  are  to  be  found  in 
Diphtheria,  Tetanus,  Streptococcus,  and  Pneumococcus. 

To  be  of  value,  antitoxins  must  be  used  as  early  as  possible, 
before  tissue  change  has  occurred  and  before  the  toxins  have,  so  to 
speak,  got  the  upper  hand.  When  the  toxins  are  in  the  ascendency 
the  patient  surfers  more  and  more  acutely,  and  may  succumb  before 
there  has  been  time  for  the  formation  in  his  own  body  of  the  neutral 
compound  of  toxin  and  antitoxin.  If  he  can  be  tided  over  the 
"crisis,"  theoretically  all  will  be  well,  because  then  his  own  anti- 
toxin will  eventually  gain  the  upper  hand.  But  in  the  meantime, 
before  that  condition  of  affairs,  the  only  way  is  to  inject  antitoxins 
prepared  in  some  animal's  tissues  whose  disease  began  at  an  earlier 
date,  and  thus  add  antitoxins  to  the  blood  of  the  patient,  early  in  the 
disease,  and  the  earlier  the  better,  for,  however  soon  this  is  done,  it 
is  obvious  that  the  toxins  begin  their  work  earlier  still.  It  should 
not  be  necessary  to  add  that  general  treatment  must  also  be  con- 
tinued, and  indeed  local  germicidal  treatment,  e.g.  of  the  throat  in 
diphtheria  and  the  poisoned  wound  in  tetanus.  Further,  in  a  mixed 
infection,  as  in  glandular  abscesses  with  diphtheria,  it  must  be  borne 
in  mind  that  the  antitoxin  is  specific,  and  may  therefore  probably 
fail  to  reduce  the  complication  which  must  be  treated  separately. 

In  the  production  of  antitoxins,  an  animal  is  required  from 
whose  body  a  considerable  quantity  of  blood  can  be  drawn  without 
injurious  effect.  Moreover,  it  must  be  an  animal  that  can  stand 


426     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 


f 


an  attack  of  such  diseases  as  diphtheria  and  tetanus.  Such  an 
animal  is  the  horse.  Now,  by  injecting  into  the  horse  (a)  living 
organisms  of  the  specific  disease,  but  in  non-fatal  doses,  or 
(b)  dead  cultures,  or  (c)  filtered  cultures  containing  no  bacteria 
and  only  toxins,  we  are  able  to  produce  in  the  blood  of  the  horse 
first  the  toxins  and  then  by  natural  processes  the  antitoxins  of  the 
disease  in  question.  The  non-poisonous  doses  of  living  organisms 
can  be  attenuated,  by  various  means.  Dead  cultures  have  not 
boen  much  used  to  produce  immunity  except  by  Pfeiffer.  In  actual 
practice  the  third  method  is  much  the  most  general,  viz.,  filtering  a 
fluid  culture  free  from  bacteria,  and  then  inoculating  this  in  ever- 
increasing  doses.  The  preparation  of  diphtheria  antitoxin  may  be 
taken  as  an  example,  but  what  follows  would  be  equally  applicable 
to  other  diseases,  such  as  tetanus : — 

1.  To  obtain  the  Toxin. — First  grow  a  pure  culture  of  the  Klebs- 
Loffler  bacillus  of  diphtheria  in  large  flasks  containing  "Loffler's 

medium,"  or  a  solution  made  by  mixing  three 
parts  of  blood  serum  with  one  of  beef  broth, 
and  adding  1  per  cent,  of  common  salt 
(NaCl)  and  1  per  cent,  of  peptone.  An 
alkaline  medium  is  necessary,  and  a  free 
supply  of  oxygen  and  the  presence  of  a 
large  proportion  of  peptone  in  the  medium 
favours  a  high  degree  of  toxicity.  The 
bouillon  must  be  glucose-free.  The  flask, 
thoroughly  sterilised  before  use,  is  now 
plugged  with  sterile  cotton-wool  and  incu- 
bated at  37°  C.  for  three  weeks.  Sterile  air 
may  be  passed  over  the  culture  periodically, 
thereby  aiding  the  growth.  After  the  lapse 
of  about  a  month  a  scum  of  diphtheria 
growth  will  have  appeared  over  the  surface  of  the  fluid.  This  is 
now  filtered  through  a  Chamberland  filter  into  sterilised  flasks,  and 
some  favourable  antiseptic  added  to  ensure  that  nothing  foreign  to 
the  toxin  shall  flourish.  The  flasks  are  kept  in  a  cool  place  in  the 
dark.  Here,  then,  we  have  the  product,  the  toxin,  ready  for  injection 
into  the  horse. 

The  power  of  the  toxins  is  estimated  by  subcutaneous  injection 
of  varying  amounts  into  a  number  of  guinea-pigs,  and  the  minimum 
lethal  dose  (M.L.D.)  is  obtained.  The  standard  M.L.D.  is  the 
smallest  amount  which  will  kill  a  250-gram  guinea-pig  in  four  days. 
According  to  Behring,  a  normal  M.L.D.  (expressed  as  D.T.N.1)  is 
'01  c.c. ;  a  toxin  of  which  '02  is  M.L.D.  will  therefore  be  expressed 
as  D.T.N/5 

2.  Immunisation  of  the   Horse. — The  general  principle   is   that 


Fio.  37.— Flask  used  for  Prepara- 
tion of  the  Toxin  of  Diphtheria. 


DIPHTHERIA  ANTITOXIN  427 

the  animal  is  treated  with  increasing  doses  of  the  particular  poison. 
The  toxins,  which  have  been  previously  tested  on  small  animals,  such 
as  rabbits  and  guinea-pigs,  are  injected  subcutaneously,  intramus- 
cularly, or  intravenously.  At  first  either  very  minute  doses  of  weak 
toxins,  or  toxins  which  had  been  modified  by  chemical  agents,  or  in 
other  ways,  are  employed.  In  the  case  of  tetanus,  in  the  early  stages 
the  toxin  is  usually  modified  by  being  treated  with  iodine.  The 
injection  of  the  toxin  may  be  followed  by  swelling  at  the  site  of 
inoculation,  loss  of  appetite,  general  malaise,  and  rise  of  temperature. 
When  these  have  passed  off  the  animal  receives  a  second,  rather 
larger  injection,  and  in  this  way  the  quantity  of  toxin  is  increased 
until  within  a  few  months  the  horse  is  capable  of  tolerating  many 
thousand  multiples  of  what  would  be  a  lethal  dose  if  given  as  a  first 
injection.  When  the  serum  has  reached  the  strength  suitable  for 
clinical  use,  blood  is  withdrawn  from  time  to  time  by  venesection. 

It  is  evident  that  only  healthy  horses  are  of  service  in  pro- 
viding healthy  antitoxin,  even  as  healthy  children  are  necessary 
in  arm-to-arm  vaccination.  To  provide  against  any  serious 
taint,  the  horse  is  tested  for  glanders  (with  mallein)  and  for 
tuberculosis  (with  tuberculin).  The  dose  of  the  injection  of 
toxin  is  at  the  commencement  about  ^  c.c.,  or  a  little  more. 
The  site  of  the  inoculation  is  the  apex  of  the  shoulder,  which 
has  been  antiseptically  cleaned.  After  the  first  injection  there  is 
generally  a  definite  febrile  reaction  and  a  slight  local  swelling. 
From  TV  or  J  c.c.  the  dose  is  steadily  increased,  until  at  the  end  of 
two  or  three  months  *  perhaps  as  much  as  300  c.c.  (or  even  half  a 
litre)  may  be  injected  without  causing  the  reaction  which  the  initial 
injection  of  y^  c.c.  caused  at  the  outset.  This  shows  an  acquired 
tolerance  of  the  tissues  of  the  horse  to  the  toxic  material.  After 
injecting  500  c.c.  into  the  horse  without  bad  effect,  the  animal  has 
a  rest  of  four  or  five  days. 

3.  To  obtain  the  Antitoxin. — During  this  period  of  rest  the 
interaction  between  the  living  body  cells  of  the  horse  and  the  toxins 
results  in  the  production  in  the  blood  of  an  antitoxin.  By  means 
of  a  small  sterilised  cannula,  five,  or  eight,  or  even  ten  litres  of  blood 
are  drawn  from  the  jugular  vein  of  the  horse  into  sterilised  flasks  or 
jars.  As  used  in  Paris,  the  top  of  the  jar  is  closed  by  two  paper 
coverings  before  it  is  sterilised.  Then  it  is  again  covered  with  a 
further  loose  one.  Before  use  the  loose  one  is  removed  and  replaced 
by  a  metal  (zinc)  lid,  which  has  been  separately  sterilised.  This 
metal  lid  contains  an  aperture  large  enough  for  the  tube  which 

*  To  shorten  this  period  Dr  Cartwright  Wood  has  adopted  a  plan  by  which  time 
may  be  saved,  and  200  c.c.  injected,  say,  within  the  first  two  or  three  weeks.  This  is 
accomplished  by  using  a  "serum  toxin"  (containing  albumoses,  but  not  ferments) 
previously  to  the  broth  toxin. 


428     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

conveys  the  blood  from  the  cannula  to  pass  through.  The  tube, 
therefore,  passes  through  the  metal  lid  and  two  paper  covers,  which 
it  was  made  to  pierce.  When  enough  blood  has  passed  into  the 
vessel  the  tube  is  withdrawn,  and  the  metal  lid  slightly  turned. 
Thus  the  contained  blood  is  protected  from  the  air.*  The  jar  con- 
taining the  blood  (which  contains  the  antitoxins)  is  next  placed  in 
a  dark,  cool  cellar,  where  it  stands  for  separation  of  the  clot.  During 
this  time  the  blood  naturally  coagulates,  the  corpuscles  falling  as  a 
dense  clot  to  the  bottom,  and  the  faintly  yellow  serum  rising  to  the 
top.  The  serum,  or  liquor  sanguinis,  averages  about  50  per  cent,  of 
the  total  blood  taken.  Sometimes  antiseptic  ("3  per  cent,  carbolic 
acid)  is  added  with  a  view  to  preservation.  It  is  generally  filtered 
(through  a  Berkefeld)  before  bottling  for  therapeutic  use,  and 
examined  bacteriologically  as  a  test  of  purity,  for  sterility  and  for 
absence  of  toxicity,  and  for  antitoxic  value. 

The  latter  step  is  the  estimation  of  the  antitoxic  power  of  the 
serum,  or  what  is  termed  the  "  standardising "  of  the  serum.  This 
is  accomplished  by  testing  the  effect  of  various  quantities  upon  a 
certain  amount  of  toxin.  Ehrlich  has  adopted  as  the  immunity  unit 
the  amount  of  antitoxic  serum  which  will  neutralise  a  hundred  times 
the  minimum  lethal  dose  of  toxin,  the  serum  and  toxin  being  mixed 
together,  diluted  up  to  4  c.c.,  and  injected  subcutaneously.  A  normal 
antitoxic  serum  is  one  of  which  1  c.c.  contains  an  immunity  unit. 

Process  of  Standardisation  of  Antitoxins. — This  matter  will  be 
best  illustrated  by  an  illustration,  as  follows : — 

Stage  1. — Varying  amounts  of  a  toxin  are  added  to  a  definite  amount  of  antitoxin, 
i.e.  to  1  Ehrlich  unit,  and  injected  into  a  guinea-pig  of  250  grammes.  That  mixture, 
which  kills  the  guinea-pig  in  four  days,  is  held  to  contain  1  M.L.D.,  over  and  above 
the  amount  of  toxin  required  to  neutralise  1  antitoxin  unit.  The  total  amount  of 
toxin  used  to  bring  about  the  death  of  the  guinea-pig  in  four  days  =  the  standard 
toxin  for  that  particular  standardisation. 

Stage  2. — Varying  quantities  of  the  antitoxin  to  be  tested  are  added  to  the 
standard  toxin  and  injected  into  guinea-pigs.  That  mixture,  which  kills  the  guinea- 
pig  in  four  days  as  before,  contains  1  M.L.D.  of  toxin  over  and  above  that 
neutralised  by  the  added  antitoxin.  The  amount  of  toxin  used  in  Stages  1  and  2 
is  the  same,  therefore  the  amount  of  antitoxin  in  Stages  1  and  2  must  be  equal.  In 
Stage  1,  1  Ehrlich  unit  was  used,  therefore  the  amount  of  antitoxin  in  Stage  2  which, 
with  the  standard  toxin  killed  the  guinea-pig  in  four  days,  also  contains  1  Ehrlich  unit. 

Example.  Stage  1. 

•25  c.c.  Toxin  + 1  Ehrlich  Antitoxin  Unit  .     Guinea-pig  alive  fifth  day. 

*— o  c.c.  ,,  ,,  ,,  .  ,,  ,, 

"27  c.c.  „  ,,  ,,  .  ,,          died  fourth  day. 

•28  c.c.  ,,  „  „  .  „          died  first  day. 

.'.  '27  c.c.  Toxin  -  Standard  toxin,  containing  1  M.L.D.  of  toxin  after  neutral- 
isation with  1  Antitoxin  unit. 

*  At  the  conclusion  of  the  operation  the  cannula  is  removed  from  the  jugular  vein 
and  the  wound  is  closed  by  the  valvular  character  of  the  slit  in  the  skin  and  vein, 
and  the  elasticity  of  the  wall  of  the  vein.  No  stitching  or  dressing  is  required. 


DIPHTHERIA  ANTITOXIN  429 

Example.  Stage  2. 

ifa  c.c.  Antitoxic  Serum  to  be  tested  +  -27  c.c.  Toxin    .     Guinea-pig  alive  fifth  day. 

•5-5-75-  C.C.  ,.  ,,  i,  •  ,,  ,, 

Tfc  C.C.  „  „  „  ,  ,,  died  fourth  day. 

^  c.c.  „  „  „  .  „  died  first  day. 

/.  The  mixture  of  ^  c.c.  of  Antitoxic  Serum +'27  c.c.  Toxin,  killing  guinea- 
pig  in  four  days,  contains  1  M.L.D. 

•'•  innr  c-c-  Antitoxic  Serum  =  1  Ehrlich  unit. 

/.   1  c.c.  =  300  Ehrlich  units. 

4.  The  Use  of  Antitoxin. — The  antitoxin  is  now  ready  for  injection 
into  the  patient  who  has  contracted  diphtheria,  and  in  whose  blood 
toxins  are  in  the  ascendency,  and  under  which  the  individual  may 
succumb.     They  are  injected  in  varying  doses,  as  we  have  already 
pointed  out.     As  large  a  dose  should  be  given  as  practicable.     A 
common  first  dose  varies  from  2000  to  5000  units.     For  prophylactic- 
purposes  a  smaller  dose  is  administered  (500,  and  for  children  under 
two   years   of   age,   300   units).     Early   administration  is  of   great 
importance.     The  flank  between  the  crest  of  the  ilium  and  the  last 
rib  and  the  lower  part  of  the  abdomen  are  generally  selected  as  the 
sites  of  injection,  but  any  region  with  loose  subcutaneous  connective 
tissue  is   suitable.      The  injections   should    be    subcutaneous.      In 
performing    the   injection    strict    asepsis  must   be   observed.     The 
syringe  must  be  well  washed  and  boiled  before  use.     The  skin  must 
be  well  cleansed  with  soap  and  water,  and  afterwards  treated  with  an 
antiseptic  such  as  a  1  in  1000  corrosive  sublimate  solution,  or  1  in 
20  carbolic  acid  solution.     The  antitoxin  of  diphtheria  has  been  used 
on  various  recent  occasions  as  a  prophylactic  in  outbreaks  of  the 
disease,  and  it  is  now  considered  as  one  of  the  practicable  means  for 
controlling  an  epidemic.     Antitoxin  inoculation  played  a  greater  or 
less  part  in   the  checking  of  diphtheria  outbreaks  at  Cambridge,* 
Colchester,-)-   Kempston,|  and    other    places.      In    the    Cambridge 
outbreak  antitoxin  was  supplied  free  for  prophylactic  use  in  the  case 
of  those  who  had  come  into  contact  with  actual  cases  of  diphtheria, 
or  where  those  who,  not  being  ill,  were  known  by  bacteriological 
examination  of  the  throat  to  be  harbouring  the  diphtheria  bacillus. 
Thus  free  bacteriological  examination  of  the  throat  of  suspected  or 
known   "contacts"   was   first   carried   out.     In   the   cases   yielding 
positive  results  antitoxin  was  injected.     At  Cambridge  500  units  of 
antitoxin  were  given  in  such  contact  persons;   at  Kempston  1000 
units  was  the  dose.     The  general  result  is  that  mortality  has  been 
lessened,  and  that  in   fatal   cases   there  has   been   a   considerable 
lengthening   of  the  period   of  life.     Moreover,  the  whole   clinical 
course  of  the  disease  is  greatly  modified,  and  its  severity  reduced. 

*  Jour,  of  Hygiene,  1901,  vol.  i.,  pp.  228  and  487. 

t  Ibid.,  1902,' vol.  ii.,  p.  170. 

$  Report  on  an  Outbreak  of  Diphtheria  at  Kempston,  p.  21. 


430     THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 

Effect  of  Diphtheria  Antitoxin  Inoculation 

The  following  summary  of  the  Antitoxin  Treatment  of  all  forms  of  Diphtheria  at 
the  Hospitals  of  the  Metropolitan  Asylums  Board,  1895-1903,  compared  with  the 
results  obtained  before  the  adoption  of  that  treatment,*  affords  striking  evidence  of 
the  efficacy  of  diphtheria  antitoxin  : — 


Cases. 

Deaths. 

Mortality, 
per  cent. 

1890-3  (before  antitoxin)  . 
1894  (antitoxin   occasion- 

7111 

2161 

30-39 

ally  used)  . 

3042 

902 

29-65 

1895  . 

2182 

615 

28-1 

1896  . 

2764 

717 

25-9 

1897  . 

4381 

896 

20-4 

1898  . 

5186 

906 

17'5 

1899  . 

7038 

1082 

15-3 

1900  . 

7271 

936 

12-8 

1901  . 

6499 

817 

12-5 

1902  . 

6015 

714 

11-8 

1903  . 

4839 

493 

10-1 

The  value  is  particularly  noticeable  among  children.  Amongst  cases  in  the  first 
year  of  life  the  rate  has  fallen  from  61-8  to  37*8;  in  the  second  year  from  63'1  to 
35-4  ;  in  the  third  year  from  55 '1  to  26 '4  ;  in  the  fourth  year  from  48 '3  to  22-9 ;  and 
in  the  fifth  year  from  39-6  to  20-7.  t 

At  the  Brook  Hospital,  Shooters  Hill,  Woolwich  (Metropolitan  Asylums  Board), 
Dr  MacCombie  has  kept  records  since  1897  showing  the  results  of  the  antitoxin 
treatment  on  all  the  cases  at  the  hospital,  with  special  reference  to  the  day  of  the 
disease  on  which  treatment  began,  in  order  to  illustrate  the  effect  of  early  adminis- 
tration : — 


Mortality  per  cent,  in  Cases  Treated. 

1897. 

1898. 

1899. 

1900. 

1901. 

1902. 

Cases  treated  on  1  st  day  of  disease 

o-o 

o-o 

o-o 

o-o 

o-o 

O'O 

»»           *i           2nd            ,, 

5-4 

5-0 

3-8 

3-6 

4-1 

4-6 

3rd 

11-5 

14-3 

12-2 

6-7 

11-9 

10-5 

4th 

19-0 

181 

20-0 

14-9 

12-4 

19-8 

»*           ,,           5th  day  and  after 

21-0 

22-5 

20-4 

21-2 

16-6 

19-4 

*  Annual  Report  of  Metropolitan  Asylums  Board,  1902,  p.  172. 

t  For  the  most  complete  account  of  diphtheria  antitoxin  and  its  effects,  see  Report 
on  the  Bacteriological  Diagnosis  and  Antitoxic  Serum  Treatment  of  Cases  admitted  to 
the  Hospitals  of  the  Metropolitan  Asylums  Board,  1895-6,  by  Professor  Sims  Wood- 
head,  M.D. 


DIPHTHERIA  ANTITOXIN  431 

"  During  the  past  six  years,"  Dr  MacCombie  reports,  **  the  total  number  of  cases 
treated  with  antitoxin  has  been  4202.  Not  a  single  death  has  taken  place  among 
the  cases  that  came  under  treatment  on  the  first  day  of  disease,  and  among  those 
coming  under  treatment  on  the  second  day  of  disease,  the  mortality  has  not  exceeded 
5-4,  and  has  been  as  low  as  3 '6.  While  among  those  that  came  under  treatment  later 
the  average  mortality  is  very  much  higher.  Were  it  possible  to  secure  the  admission 
to  hospital  of  all  cases  on  the  first  or  second  day  of  illness,  the  lives  of  a  large  number 
of  patients  would  thereby  be  saved."*  Dieudonne  has  collected  similar  returns  to 
the  foregoing  table  from  four  or  five  different  sources.  The  importance  of  early 
administration  is  therefore  widely  established. 

*  Annual  Report  of  Metropolitan  Asylums  Board,  1902,  p.  208  ;  see  also  Report, 
1903,  p.  218. 


CHAPTEE  XIII 


DISINFECTION 


General  Principles — Means  of  Disinfection:  by  Heat;  by  Chemicals— Practical 
Disinfection :  Rooms,  Walls,  Bedding,  Clothing,  Excreta,  Books,  Linen, 
Stables,  etc. — Disinfection  of  Hands— Disinfection  after  Special  Diseases  : 
Phthisis,  Small-pox,  Scarlet  Fever,  Diphtheria,  Typhoid,  Plague. 

THE  object  of  modern  bacteriology  is  not  merely  to  accumulate 
tested  facts  of  knowledge,  nor  only  to  learn  the  truth  respecting 
the  morphology  and  life-history  of  bacteria.  These  are  most 
important  things  from  a  scientific  point  of  view.  But  they  are 
also  a  means  to  an  end ;  that  end  is  the  prevention  of  preventable 
diseases  and  the  treatment  of  any  departure  from  health  due  to 
micro-organisms.  In  a  science  not  a  quarter  of  a  century  old,  much 
has  already  been  accomplished  in  this  direction.  The  knowledge 
acquired  of,  and  the  secrets  learned  from,  these  microscopic 
vegetable  cells  which  possess  such  potentiality  for  good  or  evil 
have  been,  in  some  degree,  successfully  turned  against  them.  When 
we  know  what  favours  their  vitality  and  virulence,  we  know 
something  of  the  physical  conditions  which  are  inimical  to  their 
life ;  when  we  know  how  to  grow  them,  we  also  know  how  to  kill 
them. 

We  have  previously  made  a  brief  examination  of  the  methods 
which  are  adopted  for  opposing  bacteria  and  their  products  in  the 
tissues  and  body  fluids.  We  must  now  turn  to  consider  shortly  the 
modes  which  may  be  adopted  in  preventive  medicine  for  opposing 
bacteria  outside  the  body. 

It  will  be  clear  at  once  that  we  may  have  varying  degrees  of 
opposition  to  bacteria.  Some  substances  kill  bacteria,  and  are  thus 

432 


GENERAL  PRINCIPLES  433 

germicides  ;  other  substances  prevent  their  development  and  resulting 
septic  action,  and  are  termed  antiseptics.  The  word  disinfectant  is 
used  more  or  less  indiscriminately  to  cover  both  these  terms.  ^  A 
deodorant  is,  of  course,  a  substance  removing  the  odour  of  evil- 
smelling  putrefactive  processes.  These  are  the  four  common 
designations  of  substances  able  to  act  injuriously  on  bacteria  and 
their  products  outside,  or  upon  the  surface  of,  the  body.  But  a 
moment's  reflection  will  bring  to  mind  two  facts  not  to  be  forgotten. 
In  the  first  place,  an  antiseptic  applied  in  very  strong  dose,  or  for  an 
extended  period,  may  act  as  a  germicide;  and,  vice  versa,  a 
germicide  in  too  weak  solution  to  act  as  such  may  perform  only 
the  function  of  an  antiseptic.  Moreover,  the  action  of  these  dis- 
infecting substances  not  only  varies  according  to  their  own  strength 
and  mode  of  application,  but  it  varies  also  according  to  the  specific 
resistance  of  the  protoplasm  of  the  bacteria  in  question.  Examples 
of  the  latter  are  abundant ;  for  instance,  between  the  bacillus  of 
typhoid  fever  and  the  spores  of  anthrax  there  is  an  enormous 
difference  in  power  of  resistance.  In  the  second  place,  there  are 
the  physical  conditions  injurious  to  the  development  of  bacteria. 
At  a  low  temperature  bacteria  do  not  multiply  at  the  same  rapidity 
as  at  blood-heat.  Within  the  limits  of  a  moist  perimeter  the 
air  is,  to  all  intents  and  purposes,  germ-free.  Direct  sunlight  has 
a  definitely  germicidal  effect  in  the  course  of  time  upon  some  of 
the  most  virulent  bacteria  we  know.  In  a  certain  sense  these 
three  examples  of  physical  conditions — low  temperature,  moist 
perimeter,  direct  sunlight — may  become  first  antiseptics  and  then 
germicides.  Yet  for  a  limited  period  they  have  no  injurious  effect 
upon  bacteria.  These  would  seem  to  be  very  simple  points,  and 
calling  for  little  comment,  yet  the  pages  of  medical  and  sanitary 
journals  reveal  not  a  few  keen  controversies  upon  the  injurious 
action  of  certain  substances  upon  certain  bacteria,  owing  to  the 
discrepancies  of  necessity  arising  between  results  of  different 
skilled  observers  who  have  been  carrying  out  different  experiments 
with  different  solutions  of  the  same  substance  upon  different  proto- 
plasms of  the  same  species  of  bacteria.  We  feel  no  doubt  that  in 
these  pioneering  researches  much  labour  has  been  to  some  extent 
misspent,  owing  to  the  neglect  of  a  common  denominator.  Only  a 
more  accurate  knowledge  of  bacteria  or  a  recognised  standard  for 
disinfecting  experiments  can  ever  supply  such  common  denominator. 
Species  of  bacteria  for  comparative-observation-experiments  into  the 
action  of  chemical  or  physical  agents  must  be  not  only  the  same 
species,  but  cultured  under  the  same  conditions,  and  treated  by  the 
agent  in  the  same  manner,  otherwise  the  results  cannot  be  compared 
upon  a  common  basis,  or  with  any  hope  of  arriving  at  comparable 
conclusions. 

2  E 


434  DISINFECTION 

In  1884  was  issued  from  the  English  Local  Government  Board 
one  of  the  first  adequate  statements  respecting  the  principles  of 
disinfection,  as  applied  to  the  facts  known  respecting  bacteria.* 
In  that  report  Dr  Franklin  Parsons  arrived  at  the  following 
important  conclusions:  (a)  that  all  infected  articles  which  could 
be  treated  by  boiling  water  could  not  be  so  well  disinfected  in 
any  other  way  as  by  simply  boiling  for  a  few  minutes;  (6)  that 
for  articles  which  could  not  be  so  treated,  high  pressure  steam,  with 
complete  penetration,  was  most  satisfactory ;  and  (c)  where  articles 
would  be  injured  by  either  boiling  or  steam,  dry  heat  at  240°  F.,  if 
sufficiently  prolonged,  would  be  effectual.  He  found  that  with  the 
exception  of  anthrax  spores  all  the  infected  materials  he  experimented 
upon  were  destroyed  after  an  hour's  exposure  to  dry  heat  at  220°  F., 
or  five  minutes  exposure  to  steam  at  212°  F.  Anthrax  spores 
required  four  hours'  dry  heat  at  220°  F.  Dry  heat  penetrates  very 
slowly  into  bulky  and  badly  conducting  articles,  such  as  bedding. 
Parsons  also  pointed  out  that  at  or  above  250°  F.  "scorching" 
occurred,  and  above  212°  F.  many  kinds  of  stains  were  fixed  in 
fabrics,  so  that  they  could  not  be  removed  by  washing.  He  advocated 
that  the  standard  of  true  disinfection  should  be  the  destruction  of 
the  most  stable  infective  matter  known. 

Previously  to  this  period,  experiments  had  shown  the  efficacy  of 
washing  articles  in  boiling  water,  and  Koch  had  shown  the  value  of 
corrosive  sublimate.  He  had  also  shown  the  inefficacy  of  dry  heat, 
and  of  a  number  of  chemical  substances  which  it  had  been  supposed 
were  disinfectants. 

In  1887  the  Committee  on  Disinfectants  of  the  American  Public 
Health  Association  reported  a  number  of  findings,  as  the  result  of 
experiment,  which  crystallised  known  facts.  For  infectious  material 
containing  spores  or  sporulating  bacilli  they  recommended  burning, 
steam  under  pressure  105°  C.  for  ten  minutes,  boiling  in  water  for 
thirty  minutes,  chloride  of  lime  4  per  cent.,  and  mercuric  chloride 
1-500.  If  such  material  did  not  contain  spores,  or  sporulating 
bacilli,  a  2  per  cent,  solution  of  chloride  of  lime  sufficed,  also  mercuric 
chloride  1-2000,  carbolic  acid  5  per  cent.,  chlorinated  soda  10  per 
cent.,  and  sulphur  if  3  to  4  Ibs.  per  1000  cubic  feet,  and  exposure 
not  less  than  twelve  hours.  For  excreta  the  Committee  advised 
chloride  of  lime  4  per  cent.,  for  soiled  underclothing,  bed  linen,  etc., 
burning,  boiling,  or  immersion  for  four  hours  in  mercuric  chloride 
(1-2000),  or  carbolic  acid  (2  per  cent.).  For  washing  furniture 
or  hands  the  same  solution  of  carbolic  acid;  for  disinfecting  the 
bodies  of  the  dead  carbolic  acid  (5  per  cent.),  chloride  of  lime  (4  per 
cent.),  or  mercuric  chloride  (1-500);  and  for  washing  surfaces  in 

*  Report  of  Medical  Officer  of  Local  Government  Board,  1884. 


MEANS  OF  DISINFECTION  435 

sick  rooms  and  hospitals,  2  per  cent,  carbolic,  or  1-1000  solution  of 
mercuric  chloride.* 

More  recently  a  number  of  experiments  have  been  carried  out 
in  Europe  and  America  as  to  the  efficacy  of  certain  chemical 
substances,  and  reference  will  be  made  subsequently  to  some  of  the 
results.  Much  of  the  evidence  has  been  of  a  conflicting  nature 
which  is  due,  as  we  have  said,  to  varying  conditions,  strengths  of 
disinfectants,  and  resistance  of  organisms. 

Two  or  three  years  ago  several  workers  at  Leipzig  f  drew  up 
simple  directions,  the  adoption  of  which  would  considerably  assist 
in  securing  a  common  standard  for  disinfectant  research.  They  were 
as  follows: — 

1.  In   all   comparative   observations  it  is  imperative  that  molc- 
cularly  equivalent  quantities  of  the  reagents  should  be  employed. 

2.  The  bacteria  serving  as  test  objects  should  have  equal  powers 
of  resistance. 

3.  The  number  of  bacteria  used  in  comparative  observation  should 
be  approximately  equal. 

4.  The  disinfecting  solution  should  always  be  used  at  the  same 
temperature  in  comparative  experiments. 

5.  The  bacteria  should  be  brought  into  contact  with   the   dis- 
infectant with  as  little  as  possible  of  the  nutrient  material  carried 
over.     (This  obviously  will  depend  upon  the  object  of  the  research.) 

6.  After  having  been  exposed  to  the  disinfectant  for  a  fixed  time, 
they  should  be  freed  from  it  as  far  as  possible. 

7.  They   should   then   be   returned   in   equal   numbers    to    the 
respective  culture  medium  most  favourable  to  the  development  of 
each,  and  kept  at  the  same,  preferably  the  optimum,  temperature 
for  their  growth. 

8.  The  number  of  surviving  bacteria  capable  of  giving  rise  to 
colonies  in  solid  media  should  be  estimated  after  the  lapse  of  equal 
periods  of  time.  J 

Means  of  Disinfection 

We  may  now  mention  shortly  some  of  the  commoner  methods 
and  substances  adopted  to  secure  efficient  disinfection.  They  are  all 
divisible,  according  to  Buchanan's  standard,  into  two  groups : — 

1.  Heat  in  various  forms ; 

2.  Chemical  bodies  in  various  forms. 

In  practical  disinfection  it  is  necessary  to  inhibit  or  kill  micro- 
organisms without  injury  to,  or  destruction  of,  the  substance  harbour- 

*  Sternberg's  Bacteriology,  p.  201  et  seq. 
t  Zeitschr.  f.  Hyg.  und  Inf.  Krank. ,  xxv. 

£  See  also  "  Standardisation  of  Disinfectants,"  by  Rideal  and  Walker,  Jour,  of 
Sanit.  Inst.,  1903. 


436  DISINFECTION 

ing  the  germs  for  the  time  being.  If  this  latter  is  of  no  moment, 
as  in  rags  or  carcases,  cremation  or  burning  is  the  simplest  and  most 
thorough  treatment.  But  with  mattresses  and  beddings,  bedclothes 
and  garments,  as  well  as  with  the  human  body,  it  is  obvious  that  as 
a  rule  something  short  of  burning  is  required. 

Disinfection  by  Heat 

From  the  earliest  days  of  bacteriology  heat  has  held  a  prominent 
place  as  a  means  of  disinfection.  But  it  is  only  in  comparatively 
recent  times  that  it  has  been  fully  established  that  moist  heat  is  the 
only  really  efficient  form  of  heat  disinfection.  Boiling  at  atmo- 
spheric pressure  (100°  C.  or  212°  F.)  is  the  oldest  form  of  moist  heat 
disinfection,  and  because  of  the  simplicity  of  its  application  it  has 
gained  a  large  degree  of  popularity.  But  it  must  not  be  forgotten 
that  mere  boiling  (100°  C.)  may  not  effectually  remove  the  spores  of 
all  bacilli,  and  obviously  boiling  is  not  applicable  to  furniture, 
mattresses,  and  similar  objects.  For  such  objects  hot-air  ovens  were 
used  in  former  days.  But  it  was  found  that  such  dry  heat  disinfec- 
tion (150°  C.  for  an  hour)  injured  articles  of  clothing,  etc.,  and  yet 
left  many  organisms  and  spores  untouched,  as  the  degree  of 
temperature  was  rarely,  if  ever,  uniform  throughout  the  substance 
being  treated.  The  failures  following  in  the  track  of  these  methods 
were  an  indication  of  the  need  for  some  form  of  moist  heat,  viz., 
steam. 

When  water  is  heated  certain  molecular  changes  take  place,  and 
at  a  certain  temperature  (100°  C.,  212°  F.)  the  water  becomes  steam, 
or  vapour,  and  on  very  little  cooling,  or  on  coming  into  contact  with 
cooler  bodies,  will  condense  and  give  off  its  latent  heat.  But  if  the 
vapour  is  heated,  it  will  become  practically  a  gas,  and  will  not 
condense  until  it  has  lost  the  whole  of  the  heat,  i.e.  the  heat  of 
making  water  into  vapour  plus  the  heat  of  making  vapour  into  gas. 
A  gas  proper  is,  then,  the  vapour  of  a  liquid  of  which  the  boiling 
point  is  substantially  below  the  actual  temperature  of  the  gas. 
But  we  know  that  the  temperature  at  which  it  boils  depends  on  the 
pressure  to  which  it  is  subjected  (Eegnault's  law).  Hence  in  reality 
"steam  at  any  temperature  whatever  may  be  a  vapour  proper, 
provided  the  pressure  is  such  as  prevents  the  liquid  from  boiling 
below  that  temperature."  In  such  a  condition  of  vapour  it  is  termed 
saturated  steam,  or  steam  at  or  near  its  condensation  point.  Steam 
at  any  pressure  is  "saturated,"  when  it  is  .at  the  boiling-point  of 
water  for  that  pressure.  But  if  it  is  at  that  same  pressure  further 
heated,  it  becomes  practically  a  gas,  and  is  called  superheated  steam, 
or  steam  heated  above  its  natural  condensation  point.  The  former 
can  condense  without  cooling ;  the  latter  cannot  so  condense  at  the 


BY  STEAM  437 

same  pressure.  Saturated  steam  condenses  immediately  it  meets  the 
object  to  be  disinfected,  and  gives  out  its  latent  heat;  superheated 
steam  acts  by  conduction,  and  not  uniformly  throughout  the  object. 
Its  advantage  is,  that  it  dries  moistened  objects.  It  differs  physically 
from  saturated  steam,  because  it  does  not  condense  (and  give  out  its 
latent  heat),  until  its  temperature  falls.  Therefore,  as  a  disinfecting 
power,  superheated  steam  is  much  less  than  saturated  steam,  it  has 
less  heat  in  it,  so  to  speak,  and  it  has  less  penetrative  power.  One 
further  term  must  be  defined,  namely,  current  steam.  This  is  steam 
escaping  from  a  disinfector  as  fast  as  it  is  admitted,  and  may  be  at 
atmospheric  or  higher  pressures.  The  disinfecting  temperature 
which  is  now  commonly  used  as  a  standard  is  an  exposure  to  saturated 
steam  of  115°  C.for  thirty  minutes. 

A  number  of  different  kinds  of  apparatus  have  been  invented  to 
facilitate  disinfection  at  this  standard  on  a  large  scale.  All  the 
larger  Sanitary  Authorities  are  now  supplied  with  some  form  of 
steam  disinfector,  though  many  are  not  furnished  with  high -pressure 
disinfectors.  Professor  Dele"pine  has  pointed  out  that  a  current  of 
steam  at  low  pressure  may  disinfect  completely.*  Whilst  such  simple 
current-steam  machines  have  thus  been  demonstrated  as  efficient 
bactericides,  for  practical  purposes  it  is  important  to  have  disin- 
fectors, (a)  capable  of  giving  temperatures  considerably  above  100°  C., 
(b)  of  simple  construction,  (c)  having  a  constant  steam  power  of  uni- 
form temperature  and  rapid  penetration,  and  (d)  containing,  when  in 
action,  a  minimum  of  superheated  steam.  In  addition  to  these 
characters  of  a  first-rate  steam  disinfector,  two  other  important 
points  in  actual  management  should  be  borne  in  mind,  namely,  the 
air  must  be  completely  ejected  from  the  disinfection  chamber  before 
the  results  due  to  steam  are  obtained,  and  some  sort  of  automatic 
indicator  giving  a  record  of  each  disinfection  is  indispensable. 

The  five  chief  types  of  steam  disinfectors  in  common  use  are,  the 
Washington  Lyon,  the  Goddard,  Massey,  and  Warner,  the  Equifex 
(Defines),  the  Thresh,  and  the  Eeck.f 

Washington  Lyon's  apparatus  consists  of  an  elongated  boiler 
having  double  walls,  with  a  door  at  each  end.  The  body  of  the 
apparatus  is  in  a  "jacket"  for  the  purpose  of  preventing  loss  of  heat 
and  for  "  drying  "  disinfected  articles  after  the  process.  The  whole  is 
large  enough  to  admit  of  bedding  and  mattresses,  and  generally  is  so 
arranged  that  one  end  opens  into  one  room,  and  the  other  end  opens 
into  another  room.  This  convenient  position  admits  of  inserting 
infected  articles  from  one  room  and  receiving  them  disinfected  into 

*  Jour,  of  State  Med.,  December  1897,  p.  561. 

t  Full  particulars  of  these  various  disinfectors  may  be  obtained  by  communi- 
cating with  the  makers.  Elaborate  catalogues  are  now  issued  with  illustrations 
and  details  of  working. 


438  DISINFECTION 

the  other  room.  Possible  reinfection  is  thereby  removed.  Steam 
is  admitted  into  the  jacket  at  a  pressure  of  between  20  and  25 
Ibs.,  and  its  penetrating  power  may  be  increased  by  intermitting 
the  pressure  during  the  disinfection.  At  the  end  of  the  operation 
a  partial  vacuum  is  created,  by  which  means  much  of  the  moisture 
on  the  articles  may  be  removed.  In  some  cases  a  current  of  warm 
air  is  admitted  before  disinfection  in  order  to  diminish  the  extent  of 
condensation. 

The  Equifex  (Defries)  contains  no  steam  jacket,  but  coils  of  pipes 
are  placed  at  the  top  and  bottom  of  the  apparatus,  with  the  object  of 
imparting  to  the  steam  as  much  heat  as  is  lost  by  radiation  through 
the  walls  of  the  disinfecting  chamber,  and  at  the  same  time  of  pre- 
venting undue  condensation,  and  to  be  available  for  drying.  The  air 
is  first  removed  by  a  preliminary  current  of  steam,  after  which  steam 
at  a  pressure  of  10  Ibs.  is  intermittently  introduced  (for  about 
five  minutes),  and  allowed  to  escape.  The  object  of  this  proceeding 
is  to  remove  air  from  the  pores  of  the  articles  to  be  disinfected  by 
the  sudden  expansion  of  the  film  of  water  previously  condensed  on 
their  surface. 

The  apparatus  introduced  by  Thresh  was  constructed  with  a  view 
of  overcoming  the  objection  to  some  of  the  other  machines,  that 
bulky  articles  retained  a  large  percentage  of  moisture,  thus  necessi- 
tating the  use  of  some  additional  drying  apparatus.  A  central 
chamber  receives  the  articles  to  be  disinfected,  and  is  surrounded  by 
a  boiler  containing  a  solution  of  calcium  chloride  (carbonate  of  potash 
is  now  used)  at  a  temperature  of  225°  F.  This  is  heated  by  a  small 
furnace,  and  the  steam  given  off  is  conducted  into  the  central 
chamber.  Owing  to  the  dissolved  potash  the  temperature  of  the 
steam  given  off  when  the  solution  boils  is  several  degrees  higher  than 
ordinary  steam.  The  steam  is  not  confined  under  any  pressure 
except  that  of  the  atmosphere.  When  the  steam  has  passed  for  a 
sufficient  length  of  time,  it  is  readily  diverted  into  the  open  air.  Hot 
air  is  now  introduced,  and  at  the  expiration  of  an  hour  the  articles 
may  be  taken  out  disinfected  and  as  dry  as  they  were  when  inserted. 
The  apparatus  is  comparatively  inexpensive,  and  not  of  a  complicated 
nature.  The  current  steam  is  saturated,  and  at  a  temperature  a  few 
degrees  above  the  boiling-point.  The  apparatus  is  now  made  in 
various  forms,  portable  or  otherwise. 

There  are  many  other  forms  of  steam  disinfector,  including  the 
apparatus  by  G-oddard,  Massey,  and  Warner,  the  disinfector  of  Dele- 
pine,  and  that  of  Eeck  and  others,  and  each  has  its  enthusiastic 
supporters. 


BY  CHEMICALS  439 

Disinfection  by  Chemical  Substances 

The  effects  of  chemical  substances  as  solutions,  or  in  spray  or 
gaseous  form,  upon  bacteria  have  been  observed  from  the  earliest 
days  of  bacteriology.  To  some  decomposing  matter  or  solution  a 
disinfectant  was  added  and  sub-cultures  made.  If  bacteria  continued 
to  develop,  the  disinfection  had  not  been  efficient ;  if,  on  the  other 
hand,  the  sub-culture  remained  sterile,  it  was  assumed  that  disinfec- 
tion had  been  complete.  From  such  rough-and-ready  methods  large 
deductions  were  drawn,  and  it  is  hardly  too  much  to  say  that  no 
branch  of  bacteriology  contains  such  a  mass  of  unassimilated  and  un- 
assimilable  statements  as  that  relating  to  research  into  disinfectants. 
Most  of  the  tabulated  and  recorded  results  are  conspicuous  in  having 
no  standard  as  regards  bacterial  growth.  Yet  without  such  a 
standard  results  are  not  comparable. 

Silk  threads,  impregnated  with  anthrax  spores,  were  placed  in 
bottles  containing  carbolic  acid  of  various  strengths,  and  at  stated 
periods  threads  were  removed  and  placed  in  nutrient  media,  and 
development  or  otherwise  observed.  But,  as  Professor  Crookshank 
pointed  out,  this  method  is  fallacious,  the  thread  being  still  wet  with 
the  solution  when  transferred  to  the  medium,  and  thus  the  culture 
was  modified  or  even  inhibited  altogether.*  It  is  unnecessary  for  us 
here  to  discuss  every  mode  adopted  by  investigators  in  similar 
researches.  We  may,  however,  point  out  that  the  most  approved 
methods  at  the  present  time  are  based  more  or  less  upon  two  simple 
modes  of  exposure.  In  one  a  known  volume  of  recent  broth  culture 
of  an  organism  grown  under  specified  conditions  is  used,  and  to  this 
is  added  a  measured  quantity  of  the  antiseptic.  At  stated  periods 
loopfuls  of  the  broth  and  antiseptic  mixture  are  sub-cultured  in 
fresh-sterilised  broth,  and  resulting  development  or  otherwise  closely 
observed.  The  other  method  is  practised  in  dealing  with  volatile 
bodies.  In  such  cases  a  standard  culture  is  made  of  the  organism  in 
broth  at  a  standard  temperature.  Into  this  are  dipped  small  strips 
of  sterilised  linen.  When  thoroughly  impregnated,  these  are 
removed  from  the  broth  and  subsequently  dried  over  sulphuric  acid 
in  a  vacuum  at  38°  C.  These  may  now  be  exposed  for  a  longer  or 
shorter  period  to  the  fumes  of  the  antiseptic  in  question,  and  broth 
cultures  made  at  the  end  of  the  exposure.  It  is  obvious  that  a  very 
large  number  of  modifications  are  possible  of  these  two  simple 
devices  for  testing  the  bactericidal  power  of  chemical  substances.  It 
should  be  remembered  that  here,  perhaps,  more  than  anywhere  else 
in  bacteriological  research,  careful  "control"  experiments  are 
absolutely  necessary. 

Kecently,  Ainslie  Walker  suggested  various  conditions  of  experi- 

*  Bacteriology  and  Infective  Diseases,  p.  35. 


440  DISINFECTION 

mentation  with  a  view  to  obtaining  comparable  results.  First, 
he  recommended  the  use  of  a  well-known  disinfectant  giving 
regular  and  consistent  results,  such  as  pure  phenol.  Secondly,  the 
source  and  age  of  the  culture  used  is  of  importance.  If  the 
culture  be  in  broth,  Walker  suggests  the  following  procedure: 
to  5  c.c.  of  a  twenty-four  hours'  blood-heat  culture  of  the 
organism  add  5  c.c.  of  the  dilute  disinfectant.  Shake  and  take 
sub-cultures  at  definite  intervals  in  suitable  media.  Incubate 
for  at  least  two  days  at  37°  C.  If  an  agar  culture  be  preferred, 
take  up  part  of  the  growth  on  the  point  of  a  platinum  needle,  and  dis- 
tribute it  evenly  in  sterilised  water.  The  resulting  emulsion  may  be 
used  in  place  of  the  broth  culture.  Thirdly,  Walker  emphasises  the 
importance  of  working  with  the  same  organism  for  comparable  results.* 
A  substance,  to  be  a  satisfactory  disinfectant,  should,  according 
to  Andrewes,  possess  five  characters:  (a)  it  should  be  germicidal 
within  a  reasonable  time-limit;  (&)  it  should  not  possess  chemical 
properties  which  unfit  it  for  ordinary  use ;  (c)  it  should  be  soluble  in 
water,  or  capable  of  giving  rise  to  soluble  products  in  contact  with 
the  material  to  be  disinfected;  (d)  it  should  not  produce  injurious 
effects  on  the  human  tissues ;  and  (e)  it  should  not  be  too  costly  in 
proportion  to  its  germicidal  value.f 

Mineral  acids  (nitric,  hydrochloric,  sulphuric),  especially  concen- 
trated, are  all  germicides,  but  owing  to  their  corrosive  action  their 
application  is  limited. 

A  number  of  bodies,  such  as  chloroform  and  iodoform,  have  been 
much  advocated  as  antiseptics.  The  cost  of  the  former  and  odour  of 
the  latter  have,  however,  greatly  militated  against  their  general 
adoption. 

Chloride  of  lime  is  a  powerful  disinfectant.  Professor  Sheridan 
Dele'pine  and  Dr  Arthur  Eansome  have  demonstrated  its  germicidal 
effect  as  a  solution  (1  per  cent.)  applied  directly  to  the  walls  of  rooms 
inhabited  by  tuberculous  patients.  J  Coates  confirmed  these  results 
in  houses  in  Manchester  infected  by  consumptives.  Chlorinated 
lime  ought  to  be  used  which  will  yield  not  less  than  33  per  cent,  of 
available  chlorine.  It  may  also  be  used  in  solid  form  for  decompos- 
ing matter,  excreta,  etc. 

Mercuric  chloride  (corrosive  sublimate)  has  been  an  accepted 
germicide  for  some  time.  But  the  experiments  of  Behring, 
Crookshank,  and  others,  have  proved  that  the  weaker  solutions 
(1-4000)  cannot  be  relied  upon.  This  is,  in  part,  due  to  the  fact  that 

*  Practitioner,  1902,  Ixix.,  p.  523. 

t  Many  useful  hints  and  suggestions  as  to  testing  disinfectants,  and  on  the  whole 
process  of  disinfection  will  be  found  in  Lessons  in  Disinfection  and  Sterilisation,  by 
F.  W.  Andrewes,  M.D.,  F.R.C.P.,  1903,  p.  81  et  seq.  ;  see  also  Brit.  Med.  Jour., 
1904,  ii.,  p.  13. 

£    Brit.  Med.  Jour.,  1895,  vol.  i.,  p.  353. 


BY  SULPHUR  441 

it  forms  in  albuminous  liquids  an  albuminate  of  mercury  which  is 
inactive.  Dilute  solutions  have  the  further  disadvantage  of  being 
unstable.  Various  authorities  recommend  a  solution  of  1-500  as 
a  germicide,  and  much  weaker  solutions  are  of  course  antiseptic. 
An  ounce  each  of  corrosive  sublimate  and  hydrochloric  acid  in  3 
gallons  of  water  makes  an  efficient  disinfectant. 

Potassium  permanganate  is,  of  course,  the  chief  substance  in 
Gondy's  fluid,  as  zinc  chloride  is  in  Burnett's  disinfecting  fluid.  A  5 
per  cent,  of  the  former  and  a  2J  per  cent,  of  the  latter  are  germicidal. 
Solutions  are  used  for  street-cleansing. 

Boracic  acid  is  used  as  an  antiseptic  with  which  to  wash  sore 
eyes,  or  preserve  tinned  foods  or  milk.  It  is  not  a  strong  germicide 
(it  inhibits  rather  than  kills),  but  an  unirritating  and  effective  wash. 
Many  cases  of  its  addition  to  milk  have  found  their  way  into  the 
law  courts  owing  to  cumulative  poisoning,  and  as  a  rule  its  use  as  a 
food  perservative  should  be  deprecated. 

Carbolic  acid  has  come  much  into  prominence  as  an  antiseptic 
since  its  adoption  by  Lister  in  antiseptic  surgery.  It  is  cheap, 
volatile,  and  effective.  One  part  in  40  is  antiseptic,  and  1  in  20 
germicidal.  As  a  wash  for  the  hands  the  former  is  used,  and  a 
weaker  solution  for  the  body  generally.  Carbolic  soap  and  similar 
toilette  combinations  are  now  very  common.  At  one  time  it 
appeared  as  if  corrosive  sublimate  would  take  the  place  of  carbolic 
acid  as  an  antiseptic  solution,  but  a  large  number  of  experiments 
have  confirmed  opinion  in  favour  of  carbolic.  Crookshank  found 
that  carbolic  acid,  1  in  40,  acting  for  only  one  minute,  was  sufficient 
to  destroy  Streptococcus  pyogenes,  S.  erysipelatis,  and  Staphylococcus 
pyogencs  aureus,  and  in  the  strength  of  1  in  20  carbolic  acid  completely 
sterilised  tubercular  sputum  when  shaken  up  with  it  for  one  minute. 
Klein,  Houston,  and  Gordon,  and  other  workers  have  found  a  5  per  cent, 
solution  of  carbolic  to  be  a  reliable  disinfectant  for  almost  all  bacteria. 
Cresol,  a  member  of  the  phenol  series,  is  a  good  disinfectant  and 
the  active  element  in  lysol,  Jeye's  fluid,  creolin,  izal,  and  other 
similar  substances,  which  have  been  recently  introduced  and  have 
proved  efficacious  as  disinfectants. 

Sulphurous  acid  is  one  of  the  commonest  disinfectants  employed 
for  fumigation — the  old  orthodox  method  of  disinfecting  a  room  in 
which  a  case  of  infective  disease  had  been  nursed.  It  is  evolved,  of 
course,  by  burning  sulphur.  For  each  thousand  cubic  feet  from  1 
to  5  Ibs.  of  sulphur  is  used,  and  the  walls  may  be  washed  with 
carbolic  acid.  Dr  Kenwood  carried  out  some  experiments  in  1896 
which  appeared  to  support  a  belief  in  the  disinfecting  power  of  sulphur 
fumes.*  But  he  has  since  advocated  formaldehyde  as  preferable. 
He  found  that  the  B.  diphtherice  was  not  killed  by  sulphur  though 
Brit.  Med.  Jour.,  1896  (August),  p.  439. 


442  DISINFECTION 

markedly  inhibited,  when  the  sulphurous  gas  (S0.2)  did  not 
much  exceed  "25  per  cent.  But  the  bacillus  was  killed  where  the 
sulphur  fumes  exceeded  '5  per  cent.  Both  these  results  had 
reference  to  the  S02  in  the  air  in  the  centre  of  the  room  at  a  height 
of  4  feet,  and  after  the  lapse  of  four  hours.  There  can  be  little 
doubt  that  thoroughly  fuming  a  sealed-up  room  with  sulphur  in  a 
moist  atmosphere,  and  leaving  it  thus  for  twenty-fours,  is  generally 
if  not  always,  efficient  disinfection.  Moreover,  its  simplicity  of 
adoption  is  greatly  in  its  favour.  Anyone  can  readily  apply  it  by 
purchasing  a  few  pounds  weight  of  ordinary  roll  sulphur  and  burning 
this  in  a  saucer  in  the  middle  of  a  room  which  has  had  all  its  crevices 
and  cracks  in  windows  and  walls  blocked  up  with  pasted  paper.* 
But  it  is  almost  useless  as  a  gaseous  disinfectant  unless  used  in  a 
particular  way.  The  following  seem  to  be  the  only  lines  upon  which 
anything  like  adequate  disinfection  can  be  secured  by  means  of 
sulphur : — 

1.  The  room  to  be  disinfected  must  be  effectually  sealed  up. 

2.  Not  less   than   3   Ibs.  of  sulphur  should   be  used  for  every 
1000  cubic  feet. 

3.  Twenty-four  hours  should  elapse  between  the  time  of  lighting 
the  sulphur  and  the  unsealing  of  the  room. 

4.  The  air  in  the  room  should  be  damp  during  the  process,  and 
this  may  be  achieved  by  steam,  or  spraying  the  walls  with  water,  or 
suspending  wet  blankets.     By  this  means  sulphurous  acid  is  formed, 
which  is  the  essential  part  of  the  process. 

5.  At  the  end  of  the  twenty-four  hours  the  doors  and  windows 
should  be  kept  wide  open  for  at  least  one,  and  if  possible  for  two,  days. 

6.  Furniture  and  fixtures  should,  as  far   as   possible,  be   wiped 
down  with  a  damp  cloth  soaked  in  carbolic  or  some  other  disinfectant 
solution.     Dry  dusting  or  sweeping  should  be  strongly  deprecated. 
The  walls  may  be  stripped  in  cases  where  they  are  very  dirty  or 
where  there  has  been  a  recurrence  of  a  disease.     Sulphur  fumigation 
is  not  sufficient  in  disinfection  after  consumption. 

The  conclusions  of  Dr  Novy  respecting  the  efficacy  of  sulphur 
fumes  as  a  disinfectant  may  be  added.  He  urges  that  "sulphur 
fumes  possessed  little  or  no  action  on  most  bacteria  when  in  a 
dried  state.  If,  however,  the  specimens  are  actually  wet,  they 
will  be  destroyed  except  in  the  state  of  the  resistant  forms,  such 
as  spore  stage  and  tubercle  bacilli.  For  tubercle  bacilli  or  spore- 
containing  material,  wet  or  'dry,  it  is  of  no  value.  It  can  be  used 
for  the  disinfection  of  rooms  which  have  been  infected  with 
ordinary  disease  organisms.  From  3  to  6  Ibs.  of  sulphur  must  be 
burned  in  each  1000  cubic  feet  of  space.  The  walls,  floors,  and 
articles  should  be  sprayed  with  water.  The  room  should  be  made 

*  See  also  Public  Health,  1900,  p.  438  et  seq. 


BY  FORMALIN  443 

perfectly  tight,  and  should  be  kept  closed  at  least  twenty  hours."  * 
Calmette  states  that  sulphur  vapour  under  pressure  may  be  relied 
upon  for  the  disinfection  of  ships,  etc. 

Eecently,  formalin  has  come  much  into  favour  as  a  room 
disinfectant.  Formalin  is  a  40  per  cent,  solution  in  water  of 
formaldehyde,  a  gas  discovered  by  Hofmann  in  1869.  This  gas  is  a 
product  of  imperfect  oxidation  of  methyl  alcohol,  and  may  be 
obtained  by  passing  vapour  of  methyl  alcohol,  mixed  with  air,  over  a 
glowing  platinum  wire  or  other  heated  metals,  such  as  copper  and 
silver.  Its  formula  is  CH20,  and  it  is  a  colourless  gas  with  a  pungent 
odour,  and  having  penetrating  and  irritating  properties  particularly 
affecting  the  nasal  mucous  membrane  and  the  eyes  of  those  working 
with  it.  It  is  readily  soluble  in  water,  and  in  the  air  oxidises  into 
formic  acid  (CH202).  This  latter  substance  occurs  in  the  stings  of 
bees,  wasps,  nettles,  and  various  poisonous  animal  secretions. 
Formalin  is  a  strong  bactericide  even  in  dilute  solutions,  and,  of 
course,  volatile.  Its  use  should  be  restricted  to  disinfection  of 
articles  injured  by  heat  (furs,  etc).  A  solution  of  1-10,000  is  said 
to  be  able  to  destroy  the  bacilli  of  typhoid,  cholera,  and  anthrax.  A 
teaspoonful  to  10  gallons  of  milk  is  said  to  retard  souring.  When 
formalin  is  evaporated  down,  a  white  residue  is  left  known  as 
paraform.  In  lozenge  form  this  latter  body  is  used  by  combustion 
of  methylated  spirit  to  produce  the  gas.  Hence  we  have  three 
common  forms  of  the  same  thing-— formalin,  formic  aldehyde,  para- 
form — each  of  which  yields  formic  acid,  and  thus  disinfects.  The 
vapour  cannot  in  practice  be  generated  from  the  formalin  as  readily 
as  from  the  paraform. 

By  a  variety  of  ingenious  arrangements,  formic  aldehyde  has  been 
used  by  a  large  number  of  observers  during  the  last  two  or  three 
years.  We  may  refer  to  four  modes  of  application :  1.  The  sprayer 
produces  a  mixture  of  air  and  solution  for  spraying  walls,  ceilings, 
floors,  and  sometimes  garments.  There  are  a  number  of  different 
forms  of  spraying  apparatus  such  as  the  Equifex,  the  Mackenzie, 
the  Eobertson,  etc.  2.  The  autoclave  (Trillat's  apparatus).  In  this 
apparatus  a  mixture  of  a  30-40  per  cent,  watery  solution  of 
formaldehyde  and  calcium  chloride  (4-5  per  cent.)  is  heated  under 
a  pressure  of  three  or  four  atmospheres,  and  the  almost  pure,  dry 
gas  is  conducted  through  a  tube  passing  through  the  keyhole  of  the 
door  into  the  seated-up  room.  3.  The  paraform  lamp  (the  Alformant). 
The  principle  of  this  lamp  is  that  the  hot,  moist  products  from  the 
combustion  of  methylated  spirit  act  upon  the  paraform  tablets, 
converting  them  into  gas.  4.  Lingners  apparatus  consists  of  a  ring 
boiler  in  which  steam  is  generated  and  driven  into  a  reservoir  filled 

*  Tenth  Report  of  State  of  Maine  Board  of  Health,  1898,  p.  365.  This  report 
contains  a  digest  on  the  whole  subject  of  disinfection. 


444  DISINFECTION 

with  formalin  or  glyco-formal  (30  per  cent,  formalin  with  10  per 
cent,  glycerine),  which  is  thus  vaporised  and  ejected  in  the  form 
of  a  fine  spray  through  four  nozzles.  A  room  is  thereby  speedily 
filled  with  a  dense  formalin  vapour.  After  four  hours  exposure, 
Houston  and  the  writer  found  that  B.  pyocyaneus,  Sta/phylococcus 
pyogenes  aureus,  and  various  saprophytic  organisms  were  killed.* 
Klein  and  the  writer  found  that  B.  anthracis  and  the  tubercle 
bacillus  were  killed  by  the  same  means.  Eideal  claims  that  the 
lesistant  spores  of  anthrax  may  be  killed  when  7*5  c.c.  of  formalin 
per  cubic  metre  (85  grammes  of  formaldehyde  per  1000  cubic  feet) 
are  vaporised  with  not  less  than  four  times  its  volume  of  water,  and 
that  exposure  need  not  exceed  six  hours.f  Klein,  Houston,  and 
Gordon  found  that  B.  typhosus,  B.  dipMJierice,  and  certain  suppu- 
rative  organisms  were  killed  by  means  of  the  alformant  lamp 
method.!  It  is  agreed  that  the  gas  is  harmless  to  colours,  metals, 
leather,  and  polished  wood.  The  vapour  acts  best  in  a  warm 
atmosphere.  As  for  its  action  on  bacteria,  it  may  be  said  that  it 
compares  favourably  with  any  other  disinfectant. 

Many  observers  have  not  recommended  formaldehyde  on  account 
of  its  professed  lack  of  penetrating  power.  Professor  Delepine, 
however,  states  that  it  possesses  "penetration  powers  probably 
greater  than  those  of  most  other  active  gaseous  disinfectants.  B. 
coli,  B.  tuberculosis,  B.  pyocyaneus,  and  Staphylococcus  pyogenes  aureus 
were  killed  in  dry  or  moist  state,  even  when  protected  by  three 
layers  of  filter-paper."  §  In  Professor  Delepine's  opinion,  the 
vapours  of  phenol,  izal,  dry  chlorine,  and  sulphurous  acid  have, 
under  the  same  conditions,  given  inferior  results.  Since  1898  a 
number  of  experimenters  have  confirmed  these  opinions.  It  is 
extremely  important  that  that  disinfectant  should  be  used  which  is 
the  most  suitable  one  for  the  particular  purpose  at  issue.  A 
germicidal  substance  which  under  certain  conditions,  and  in  relation 
to  one  species  of  organism  may  be  practically  useless,  may  under 
other  conditions  be  most  efficacious. 


Practical  Disinfection  II 

To  disinfect  a  room,  seal  up  cracks  and  crevices,  spray  the  walls 
with    water,   and    burn,   say,   3-6   Ibs.    of   roll   sulphur   for   every 

*  Practitioner,  1902,  vol.  Ixix.,  p.  328. 

t  Jour,  of  Sanitary  Institute,  1903,  vol.  xxiii.,  part  iv. 

£  Report  of  Medical  Officer  of  London  County  Council,  1902. 

§  Jour,  of  State  Med.,  1898  (November),  p.  541. 

II  For  hints  in  the  detail  management  of  disinfection,  the  reader  is  recommended 
to  study  A  Practical  Guide  to  Disinfection,  by  Rosenau  and  Allan,  1903 ;  Lessons 
in  Disinfection,  by  F.  W.  Andrewes,  1903;  the  Practitioner,  1902,  p.  300  (Houston); 
Rideal's  Disinfection  and  Disinfectants,  1904  ;  and  Public  Health,  1904,  pp.  558-570. 


PRACTICAL  DISINFECTION  445 

1000  cubic  feet  of  space.*  Let  the  room  remain  sealed  up  for 
twenty-four  hours,  then  be  freely  opened.  Formaldehyde  gaseous 
disinfection  may  be  used  as  described  above.  But  it  would  appear 
that  neither  sulphur  or  formaldehyde  are  always  reliable  in  dis- 
infecting after  tuberculosis.  The  most  important  point  is  to 
cleanse  surfaces,  and  probably  the  most  efficient  disinfection  of  a 
room  is  by  Lingner's  apparatus  (glyco-formal,  1  litre  to  every 
1000  cubic  feet),  coupled  with  spraying  or  washing  surfaces  with 
germicidal  solution. 

To  disinfect  walls,  floors,  etc.,  wash  or  spray  with  chloride  of 
lime  solution  (1-100),  izal  (1-100),  formalin  (2-100),  or  carbolic  acid 
(1-40).  The  last-named  solution  may  be  used  to  wipe  down  furniture. 
These  disinfectants  may  be  used  after  sulphur  fuming.  Formic 
aldehyde  may  also  be  used  by  autoclave  or  Lingner's  apparatus. 

To  disinfect  bedding,  etc.,  the  steam  sterilisation  secured  in  an 
efficient  apparatus  is  the  best  (115°  C.  for  thirty  minutes).  Eags  and 
infected  clothing,  unless  valuable,  should  be  burnt. 

To  disinfect  garments  and  wearing  apparel. — If  possible,  steam  in 
an  efficient  steriliser ;  if  that  be  not  available,  such  articles  should 
be  washed  in  a  disinfectant  solution  (5  per  cent,  carbolic),  or  fumed 
with  formic  aldehyde  (Lingner's  glyco-formal  apparatus). 

To  disinfect  excreta  or  putrefying  solutions,  enough  disinfectant 
should  be  added  to  produce  in  the  solution  or  matter  being  disinfected 
the  percentage  of  disinfectant  necessary  to  act  as  such.  Adding  a 
small  quantity  of  antiseptic  to  a  large  volume  of  fluid  or  solid  is  as 
useless  as  pouring  a  small  quantity  of  antiseptic  down  a  sewer  with 
the  idea  that  such  treatment  will  disinfect  the  sewage.  The  mixture 
of  the  disinfectant  with  the  matter  to  be  disinfected  must  contain 
the  standard  percentage  for  disinfection.  Chloride  of  lime  is  a 
common  substance  for  use  in  this  way  (J  Ib.  to  a  gallon  of  water)  or 
in  a  4  per  cent,  solution.  Potassium  permanganate  (1-100),  and 
carbolic  (5  per  cent.),  and  many  manufactured  bodies  containing 
them,  are  also  widely  used.  Corrosive  sublimate  (1-500),  izal  (1-100), 
copper  sulphate  (1-20),  lysol,  cresol,  or  creolin  (1-40),  have  all  been 
found  efficacious  (Houston).  Drs  Hill  and  Abram  recommend  that 
the  excreta  and  disinfectant  be  thoroughly  mixed,  and  stand  for  at 
least  half  an  hour.")*  For  various  reasons  they  particularly  advise 
cliinosol  as  the  most  convenient  disinfectant  for  this  specific  purpose. 
But  subsequent  experience  has  perhaps  hardly  supported  this 
recommendation. 

Antiseptics  for  wounds. — Carbolic  acid  (1-40)  or  corrosive  sub- 
limate (1-1000)  are  commonly  used  in  surgical  practice.  Boracic 

::  The  measurement  of  cubic  space  is,  of  course,  made  by  multiplying  together  in 
feet  the  length,  breadth,  and  height  of  a  room. 
t  Brit.  Med.  Jour.,  1898  (April),  p.  1013. 


446  DISINFECTION 

acid  is  one  of  the  most  unirritating  antiseptics  which  is  known.  It 
may  be  used  in  saturated  watery  solution  (1-30)  or  dusted  on 
copiously  as  fine  powder.  It  is  especially  applicable  to  open  wounds, 
and  as  an  eye-wash. 

Boots,  looks,  leather-covered  articles,  etc.,  should  be  disinfected  by 
dry  heat  or  formalin  (preferably  Lingner's  apparatus). 

Infected  linen  should  be  steamed  or  boiled,  but  if  that  is  not 
available,  immersion  for  one  hour  in  corrosive  sublimate  (1-500)  or 
for  twenty-four  hours  in  the  same  solution  1-1000.  Less  powerful 
germicides  have,  however,  been  found  successful,  e.g.  izal  (1-100), 
carbolic  acid  (1-100). 

Cups,  saucers,  plates,  spoons,  knives,  forks,  etc.,  should  all  be 
disinfected  in  boiling  water. 

Rags  in  bales  can  only  be  disinfected  by  steam. 

Pens,  lyres,  stables  t  trucks,  vans,  markets,  etc.,  are  best  treated 
with  some  form  of  sprayer  (e.g.  Equifex  hot-spray  disinfector)  or 
distributor  (e.g.  the  chloros  distributor).  Ships  also  may  be  treated 
by  this  apparatus  or  by  means  of  the  Newcastle  disinfecting  hulk 
(Goddard,  Massey,  and  Warner). 

Disinfection  of  the  Hands. — To  a  surgeon,  the  disinfection  of  the 
hands  is  a  matter  of  vital  importance.  There  are  many  opportunities 
for  conveying  bacteria  on  the  hands,  which  naturally  come  in  the 
way  of  dust  and  dirt,  and  so  carry  organisms  in  the  cracks  of  the 
skin  surface,  in  the  sebaceous  glands,  under  the  nails,  and  even  in 
the  substance  of  the  epithelium.  This  was  demonstrated  by 
Lockwood  in  1896,  and  again  by  Freeman  in  1899.  Subsequent 
experiments  confirmed  the  fact  of  the  difficulty  of  completely  freeing 
the  skin  of  the  hands  from  micro-organisms.  In  1902,  Dr  Schaeffer 
of  Berlin,  whilst  recognising  that  absolute  asepticism  of  the  hands 
is  not  possible,  showed  by  experiments  that  it  is  possible  to  render 
the  hands  so  free  from  organisms  during  a  surgical  operation  that 
the  danger  of  wound  contamination  is  exceedingly  small.  Collins 
has  pointed  out  (1904)  that  much  depends  upon  vigorous  scrubbing, 
clean  nail-brushes,  and  hot  water.  Soap,  water,  and  carbolic  acid 
(1-20),  permanganate  of  potash,  corrosive  sublimate  (1-1000), 
lysol,  and  many  other  similar  antiseptic  solutions  have  been 
used  with  more  or  less  satisfactory  results.  Schaeffer,  how- 
ever, decides  in  favour  of  the  hot-water-alcohol  method  (96  per 
cent,  spirit),  the  chief  advantage  of  which  is  that  it  removes 
organisms  from  the  skin  rather  than  killing  them  on  the  skin. 
Mikulicz  advocates  spirit-soap  as  cheaper  than  alcohol,  but  apparently 
the  difference  in  expense  in  this  country  is  not  great,  and  the  spirit/- 
soap leaves  the  hands  in  a  slippery  condition.  It  may '  be  pointed 
out  that  washing  in  spirit  rather  than  antiseptics  preserves  the 
smooth  surface  of  the  skin  and  results  in  no  roughness. 


PRACTICAL  DISINFECTION  447 

Disinfection  in  or  after  Special  Diseases 

Disinfection  after  Phthisis.— The  following  statement  was 
drawn  up  in  1901,  by  Drs  Newsholme,  Mven,  and  the  writer,  for 
the  National  Association  for  the  Prevention  of  Consumption  and 
other  forms  of  Tuberculosis.  It  may  serve  as  a  basis  for  practical 
disinfection  of  rooms,  etc.,  after  phthisis : — 

"  The  necessity  for  disinfection  in  consumption  is  based  on  well- 
established  facts.  The  essential  cause  of  consumption  and  of  all 
other  forms  of  tuberculosis  is  a  living  microbe,  the  B.  tuberculosis, 
though  the  condition  of  the  bodily  health  of  the  individual  greatly 
influences  the  resistance  to  the  disease  and  the  prospect  of  recovery 
from  it. 

"  The  disease  is  always  contracted  by  taking  into  the  system  the 
microbes  causing  it,  which  are  derived  solely  from  persons  or 
animals  suffering  from  the  same  disease.  These  microbes  may  be 
taken  in  infected  milk  or  less  commonly  in  infected  flesh. 

"The  most  frequent  source  of  infection,  however,  is  the  dis- 
charges and  particularly  the  phlegm  (spit  or  expectoration)  of  a 
consumptive  person.  These  discharges  whilst  moist  are  not  likely 
to  be  scattered,  but  if  allowed  to  dry  they  become  broken  up  into 
dust,  and  are  then  extremely  dangerous.  There  is  little  or  no  risk 
of  contracting  consumption  directly  from  the  breath  of  a  consumptive 
person,  but  the  phlegm  infects  everything  upon  which  it  falls — 
handkerchiefs,  books,  papers,  linen,  floors,  carpets,  furniture,  etc., 
and  is  then  readily  inhaled  by  healthy  persons.  This  is  the  chief 
means  by  which  consumption  is  spread  from  person  to  person. 

"  On  these  facts  rest  the  important  question  of  disinfection.  In 
preventing  a  consumptive  person  from  spreading  the  disease,  two 
sets  of  preventive  measures  are  required: — 1st,  the  removal  or 
destruction  of  the  infective  matter  already  disseminated  by  the 
patient's  discharges,  especially  by  his  phlegm;  and,  2nd,  the 
prevention  of  future  dissemination.  For  the  latter  purpose  the 
main  object  is  not  to  permit  any  discharge  to  become  dry  before 
being  destroyed.  Before  the  consumptive  person  has  learned  the 
personal  precautions  which  must  be  taken,  and  up  to  the  time  when 
he  has  been  trained  to  carry  them  out  carefully,  he  has  probably 
distributed  a  considerable  amount  of  infective  matter.  This  is 
especially  liable  to  accumulate  in  a  dangerous  form  at  home,  where 
the  space  is  small,  and  light  and  ventilation  are  defective.  Infective 
particles  will  be  found  in  greatest  abundance  on  and  near  the  floors, 
on  ledges,  and  in  room-hangings.  But  the  personal  clothing  and 
bedclothes  will  also  have  become  infected.  Hence  it  is  necessary 
to  disinfect  the  floor,  walls,  and  ceiling  of  the  rooms  occupied  by 
the  patient,  as  well  as  the  furniture,  carpet,  bedclothes,  etc. 


448  DISINFECTION 

"  When  this  has  been  done,  if  the  personal  precautions  advised 
are  carried  out  by  the  consumptive,  further  disinfection  should  not 
be  needed. 

"  It  is,  however,  difficult  to  make  sure  that  personal  precautions 
are  fully  carried  out,  and  rooms  should  therefore  be  subsequently 
cleaned  at  least  once  in  six  months,  the  floors  being  scrubbed  with 
soft  soap,  the  furniture  washed,  the  walls  cleaned  down  with  dough, 
and  the  ceiling  whitewashed. 

"  Confined  workshops  in  which  a  consumptive  has  worked  for 
some  time  should  be  cleansed,  and  a  notice  in  reference  to  spitting 
should  be  suspended  in  all  workshops.  The  latter  precaution  should 
also  be  observed  in  all  public-houses  and  common  lodging-houses, 
both  of  which  require  special  attention  to  cleansing. 

"Disinfection  of  rooms  which  have  been  occupied  by  con- 
sumptive patients  may  be  secured  in  various  ways,  but  the  following 
are  the  practical  rules  which  must  underlie  any  methods  adopted : — 

"  1.  G-aseous  disinfection  of  rooms,  or  '  fumigation '  as  it  is 
termed,  by  whatever  method  it  is  practised,  is  inefficient  in  such 
cases. 

"  2.  In  order  to  remove  and  destroy  the  dried  infective  discharges, 
the  disinfectant  must  be  applied  directly  to  the  infected  surfaces  of 
the  room. 

"3.  The  disinfectant  may  be  applied  by  washing,  brushing,  or 
spraying. 

"  4.  Amongst  other  chemical  solutions  used  for  this  purpose,  a 
solution  of  chloride  of  lime  (1  to  2  per  cent.)  has  proved  satisfactory 
and  efficient. 

"  5.  In  view  of  the  well-established  fact  that  it  is  the  dust  from 
dried  discharges  which  is  chiefly  infective,  emphasis  must  be  laid 
upon  the  importance  of  thorough  and  wet  cleansing  of  infected 
rooms. 

"6.  Bedding,  carpets,  curtains,  wearing  apparel,  and  all  similar 
articles  belonging  to  or  used  by  the  patient,  which  cannot  be 
thoroughly  washed,  should  be  disinfected  in  an  efficient  steam 
disinfector. 

"  7.  After  all  necessary  measures  of  disinfection  have  been  carried 
out,  the  essential  principle  governing  the  subsequent  control  of  a 
case  of  consumption  is  that  all  discharges,  of  whatever  kind  (especi- 
cially  expectoration  from  the  lungs),  should  under  no  circumstances 
be  allowed  to  become  dry." 

In  Manchester  and  other  places,  where  disinfection  after  phthisis 
is  regularly  practised,  a  solution  of  chlorinated  lime  of  the  strength 
of  1J  ounces  to  the  gallon  is  used.  The  wall-paper  is  thoroughly 
saturated  with  this  solution,  applied  with  a  soft  brush  or  spray, 


AFTER  SPECIAL  DISEASES  449 

and  is  then,  where  necessary,  stripped  from  the  walls.  The  bare 
walls,  the  ceiling,  and  floor  are  washed  over  several  times  with 
the  solution,  and  any  articles  of  furniture  which  will  admit  of  such 
treatment  are  similarly  washed  over.  Articles  of  clothing,  bedding, 
etc.,  are  taken  away  to  be  disinfected  in  the  steam  disinfector. 

In  houses  in  Manchester  which  are  in  a  clean  condition,  and 
where  it  is  certain  that  there  has  been  no  direct  soiling  of  the  walls 
or  floors  with  sputum,  and  where  the  infectious  dust,  if  present,  has 
come  from  soiled  pocket-handkerchiefs  or  articles  of  clothing,  the 
chlorinated  lime  method  of  disinfection  is  not  considered  necessary, 
and  the  method  of  disinfection  recommended  by  Esmarch  is 
practised: — The  wall-paper  is  rubbed  well  with  crumb  of  bread,  or 
with  dough  kneaded  to  a  proper  consistency.  Floors,  painted  walls, 
and  woodwork  are  washed  with  soap  and  water,  and  ceilings  are 
limewashed.  In  addition,  bedding,  articles  of  clothing,  etc.,  are 
either  disinfected  by  steam  or  washed  with  boiling  water. 

This  method  of  disinfection,  when  properly  carried  out,  was 
found  to  remove  practically  all  dust  from  a  room,  so  that  little  or 
no  dust  can  be  obtained  by  subsequently  rubbing  the  wall-paper 
with  a  sterilised  sponge.  The  method,  however,  requires  a  certain 
amount  of  care  to  make  sure  that  all  dust  is  removed  from  the  walls, 
especially  from  the  angles  and  corners,  and  to  properly  rub  down  a 
fair-sized  room  takes  a  considerable  time.  It  is  useless  in  cases 
where  the  paper  is  directly  soiled  with  sputum.  Owing  to  the 
mucus  which  it  contains,  the  dried  sputum  sticks  tenaciously  to  the 
paper,  in  spite  of  repeated  rubbing  with  dough. 

This  method  of  rubbing  the  walls  with  dough  is  an  excellent 
way  of  periodically  cleaning  a  room,  so  as  to  keep  it  free  from 
dust. 

After  Small-Pox. — It  is  necessary  that  disinfection  be  very 
thoroughly  done.  As  a  rule,  the  walls  of  the  room  used  by  the 
patient  must  be  "  stripped  and  cleansed."  Fumigation  with  formic 
aldehyde  and  vigorous  spraying  of  walls  are  usual.  All  bedding  and 
wearing  apparel  must  be  steamed,  and  if  very  unclean,  burnt. 

After  Scarlet  Fever. — The  room  used  by  the  patient  should  be 
disinfected  in  the  ordinary  way.  Infection  may  be  conveyed  by 
clothing,  carpets,  table-cloths,  bell-ropes,  etc.,  and  such  things  must 
receive  attention.  Infection  is  also  probably  conveyed  by  the  peeling 
skin,  and  even  more  so  by  the  throat  secretions.  All  discharges 
from  the  mouth  and  nose,  and  also  those  from  the  ear  when  affected, 
should  be  received  on  rags  or  thin  paper  handkerchiefs  and  burned. 
The  seat  of  infection  may  also  be  directly  attacked  by  the  use  of 
disinfectant  gargles,  of  which  chlorine  water  is  one  of  the  best. 
During  desquamation  the  skin  may  be  oiled,  and  occasionally  washed 
in  warm  carbolic  solution  (1-40). 

2  F 


450  DISINFECTION 

After  Diphtheria. — The  bacillus  of  diphtheria  is  non-sporulating, 
and  has  comparatively  little  resistance  against  disinfectants.  Ordi- 
nary means  of  disinfection  are  therefore  sufficient.  Local  disinfec- 
tants should  be  used  for  the  throat  until  bacteriological  examination 
is  negative  to  the  Klebs-Loffler  bacillus,  which  may  persist  in  the 
throat  for  long  periods.  The  throat  may  be  painted  with  a  solution 
of  perchloride  of  mercury  (1-500);  15  to  20  minims  of  such  a 
solution  would  be  a  suitable  amount  to  use  for  a  single  application. 
Gargles  or  sprays  may  be  employed,  consisting  of  chlorine  water,  or 
permanganate  of  potash  (1-300).  The  throat  and  nose  discharges 
should  be  received  on  rags  which  can  be  burned. 

After  Typhoid  Fever  and  Cholera.— Bedding  and  articles 
which  have  come  into  contact  with  the  patient  require  attention 
in  typhoid  fever  and  cholera.  The  disinfection  of  the  excreta 
(faeces  and  urine)  is  the  most  important  item.  These  discharges 
should  not  be  passed  into  the  house  drains  until  disinfected. 
They  should  stand  for  some  hours  thoroughly  mixed  with  the 
disinfectant  before  being  considered  disinfected.  Chloride  of  lime 
(1-500  of  the  total  mixture),  izal  (1-200  of  the  total  mixture)  and 
carbolic  acid  (1-40  of  the  total  mixture)  are  all  used  in  this  way. 
If  there  is  no  house-drainage  or  water-carriage  system,  the  excreta 
should  be  treated  as  above,  and  deeply  buried  remote  from  any  well 
or  water-course.  The  nurse's  hands  must  be  kept  thoroughly 
cleansed  (thorough  washing  with  hot  water,  soap,  and  perchloride 
solution,  1-1000),  especially  before  meals. 

After  Plague. — The  detailed  arrangements  for  the  removal  of 
cases  and  disinfection  of  infected  tenements  after  plague  should  be 
under  the  personal  supervision  of  the  medical  staff,  and  may  be 
detailed  as  follows : — 

(a)  Kemoval  of  patient  to  hospital. 

(b)  Eemoval  of  "contacts"  to  reception  house,  and  kept  under 
medical  observation  for  fourteen  days. 

(G)  Fumigation  of  infected  house  by  liquefied  sulphur  dioxide  or 
formic  aldehyde  from  twelve  to  twenty-four  hours,  the  disinfectant 
being  used  in  proportion  to  the  cubic  space  dealt  with. 

(d)  After  the  fumigation  the  house  is  entered ;   all  articles  of 
clothing,  etc.,  to  be  removed  are  first  of  all  thoroughly  wetted  with 
2   per   cent,   solution  of   formalin   (1   gallon   40-per-cent.   solution 
formaldehyde  to  50  gallons  water),  or  2  per  cent,  chloride  of  lime, 
then  wrapped  up  in  sheets  soaked  in  the  same  fluid  and  removed  to 
the  sanitary  wash-house.     There  all  articles  which  cannot  be  boiled 
or  steamed,  or  treated  with  formaldehyde,  are  burned. 

(e)  The  walls,  ceiling,  flooring,  woodwork,  etc.,  and  furniture  of 
the  infected  house  are  also  sprayed  with  the  formalin  solution  (1 
gallon  to  50  gallons  water)  or  chloride  of  lime. 


AFTER  SPECIAL  DISEASES  451 

(/)  All  rooms  in  the  infected  dwelling  are  cleansed;  the  lobbies, 
stairs,  and  landings  being  dealt  with  by  formaldehyde  or  chloride  of 
lime  solution. 

(17)  Courts  of  such  dwellings  are  watered  with  chloride  of  lime 
solution. 

(h)  Ash-pits  have  contents  watered  with  same,  and  then  removed 
and  burned. 


APPENDIX 


NOTES   ON  TECHNIQUE 

Synopsis  of  Technique: — General  Methods  of  Examination;  Staining  Methods; 
Flagella;  Spores,  etc. — Bacteriological  Diagnosis — Examination  of  Water — 
Examination  of  Milk  —  Bacteriological  Diagnosis  in  Special  Diseases  — 
Examination  of  Malaria  Blood — Examination  of  Oysters — Examination  of 
Sewage — Miscellaneous. 

GENERAL  ELEMENTARY  METHODS  OF  EXAMINATION 

WITH  the  exception  of  pathological  tissue  and  similar  insoluble 
substances,  the  common  practice  in  bacteriology  is  to  reduce  as  far  as 
possible  the  article  to  be  examined  to  a  fluid,  that  is  to  say,  it  is 
chiefly  fluids  which  can  be  systematically  examined  by  the  methods 
of  bacteriology.  Water,  milk,  sewage,  urine,  blood,  etc.,  are  at  once 
in  a  condition  to  make  examination  available,  but  cheese,  butter,  foods, 
soil,  pus,  dust,  etc.,  require  to  be  reduced  to  fluid,  or  washed  in  fluid 
media,  preparatory  to  examination.  Thus  soil  particles  may  be  washed 
and  macerated  in  sterilised  broth,  and  the  broth  examined  for  contained 
organisms.  It  will,  on  this  account,  be  most  convenient  in  the  first 
place  to  consider  the  application  of  bacteriological  methods  to  the 
examination  of  fluids. 

The  principle  underlying  the  ordinary  technique  is  the  solidification 
of  fluid  gelatine  at  or  below  room  temperature.  If  a  drop  of  con- 
taminated water,  for  example,  be  added _^ 

to    a    tube    of    10    c.c.    of  liquid    gelatine,  ~~ 

thoroughly  mixed,  and  then    the  contents 

of  the  tube  poured  out  into  a  Petri   plate 

(or  other  shallow  glass  dish)  and  allowed  to  FlG  38-_petri  Dish 

solidify,   we  shall   have    scattered  through 

the  solid  film  of  gelatine  the  contained  bacteria,  in  a  favourable  medium 

for  their  growth  and    multiplication.     Such    a  plate  will  be  protected 

from  the  air  and  incubated  at  a  regulated  temperature.      This  is   the 

principle  of  Koch's  Plate  Method.     In  the  course  of  two  or  three  days 

the   film    of  gelatine    on    the   plate  becomes    covered  with   colonies   of 

germs,  consisting  of  countless  individual  bacteria  gathered  round   the 

453 


454 


APPENDIX 


parent  organism  which  found  its  way  thither  from  the  drop  of  con- 
taminated water.  The  next  step  is  to  examine  these  quantitatively 
and  qualitatively. 

1.  Naked-Eye  Observation  of  the  Colonies. — By  this  means,  at  the  very 
outset  certain  facts  may  be  obtained,  viz.,  the  size,  elevation,  configura- 
tion, margin,  colour,  grouping,  number,  and  kinds  of  colonies,  all  of 
which  facts  are  of  importance,  and  assist  in  final  determination  as  to  the 
quantity  and  character  of  the  organisms  present  in  the  original  drop  of 


Fio.  39.— A  Diagram  of  Colonies  of  Bacteria  on  a  Gelatine  Plate. 

water.  Moreover,  in  the  case  of  gelatine  medium  (owing  to  the  fact 
that  it  is  liquefiable  by  ferments),  one  is  able  to  observe  whether  or  not 
there  is  present  what  is  termed  liquefaction  of  the  gelatine.  Some 
organisms  produce  in  their  development  a  peptonising  ferment  which 
breaks  down  gelatine  into  a  fluid  condition.  Many  have  not  this  power, 
and  hence  the  characteristic  is  used  as  a  diagnostic  feature. 

2.  The  Microscopic  Examination  of  Colonies  (under  low  magnification, 
x  60-100)  confirms  or  corrects  that  which  has  been  observed  by  the  naked 
eye.  Micro-organisms,  when  growing  in  colonies,  produce  cultivation 
features  which  are  peculiar  to  themselves  (especially  is  this  so  when 


APPENDIX  455 

growing  in  test-tube  cultures),  and  in  the  early  stages  of  such  growths 
a  low  power  of  the  microscope  or  a  magnifying  glass  facilitates 
observation. 

3.  The  Making  of  Cover-glass  Preparations  :  («)  unstained — "  the  hang- 
ing drop  "  ;  (6)  stained — single  stains,  e.g.,  gentian-violet,  methylene- 
blue,  fuchsin,  carbol-fuchsin,  etc. ;  or  double  stains  by  Gram's  method, 
by  Ziehl-Neelsen's  method,  etc.  This  third  part  of  the  investigation  is 
obviously  to  prepare  specimens  for  examination  under  the  microscope.* 
"  The  hanging  drop  "  is  a  simple  plan  for  securing  the  organisms  for 

microscopic  examination  in  a  more 

or  less  natural  condition.     A  hollow 


w     K 

ground    slide     (i.e.    a    slide  with    a       \^ 
shallow  depression  in  it)  is  taken, 
and  a  small  ring  of  vaseline  placed 
round  the   edge  of  the  depression. 


Upon   the    under-side    of    a    clean  FIG.  40.-The  Hanging  Drop. 

cover-glass  is  placed  a  drop  of  dis- 
tilled water,  and  this  is  inoculated  with  the  smallest  possible  particle 
taken  from  one  of  the  colonies  of  the  gelatine  plate  on  the  end  of  a 
sterilised  platinum  wire.  The  cover-glass  is  then  placed  upon  the  ring 
of  vaseline,  and  the  drop  hangs  into  the  space  of  the  depression.  Thus 
is  obtained  a  view  of  the  organisms  in  a  freely  moving  condition,  if 
they  happen  to  be  motile  bacteria.  In  ordinary  practice  the  hollow 
slide  may  be  dispensed  with,  and  an  ordinary  slide  used. 

With  regard  to  staining,  it  will  be  undesirable  here  to  dwell  at  length 
upon  the  large  number  of  methods  which  have  been  adopted.  The 

*  A  good  microscope  is  essential.  It  should  have  objectives  of  1  inch,  £,  and 
-rV  (oil  immersion).  A  white  light,  and  proper  adjustment  of  the  substage  condenser 
and  draw-tube  are  also  necessary.  A  lens  of  TVth  inch  focal  depth  is  the  usual 
power  required  for  the  study  of  bacteria,  although  in  some  cases  a  lens  of  a  focal 
length  of  ^th  inch,  or  even  stronger,  is  desirable.  Streptothrix  actinomyces,  which 
belongs  to  the  higher  bacteria,  is  better  seen  with  a  power  of  £th  inch  than  with  one  of 
T\th  inch.  The  principle  of  the  immersion  lens  is  the  filling  up  of  the  space  between 
the  lens  and  the  cover-glass  with  a  material  whose  refractive  index  is  the  same  as 
that  of  the  lens,  so  that  there  will  be  no  loss  of  illumination  by  the  rays  of  light 
passing  through  media  of  different  powers  of  refraction,  while  proceeding  from  the 
object  to  the  lens.  The  power  of  a  microscope  varies  not  only  according  to  that  of 
the  lens,  but  also  according  to  the  power  of  the  eye-piece.  Thus  the  magnifying 
power  of  a  1-inch  objective  in  Swift's  microscope  varies,  according  to  the  strength 
of  the  eye-piece  and  to  the  fact  that  the  draw-tube  is  closed  or  extended,  from  25 
to  140  diameters ;  a  £th  inch  objective,  from  175  to  690  diameters  ;  and  a  T^th  inch 
objective,  from  385  to  1627  diameters.  As  a  high-power  lens  gives  a  picture  which 
has  comparatively  very  little  "depth"  of  focus,  it  is  necessary  to  place  the  object 
under  examination  in  as  nearly  the  same  plane  as  possible.  Hence  the  material 
to  be  investigated  should  be  reduced  to  an  extremely  thin  film. 

The  object  should  also  be  in  the  optical  axis  of  the  instrument,  and  secured  in 
position  by  means  of  the  spring  clips.  In  using  the  oil  immersion  lens,  the  body 
tube  of  the  microscope  must  be  screwed  down  until  the  lens  is  in  contact  with  the 
oil  and  nearly  touching  the  coverslip.  The  substage  condenser  must  be  screwed  up 
flush  with  the  stage.  The  best  light  must  be  obtained  by  adjustment  of  the  mirror, 
and  fine  focus  must  be  used.  A  skilful  use  of  the  microscope  depends,  of  course, 
upon  an  understanding  of  its  parts  and  upon  practice. 


456  APPENDIX 

"  single  stain "  may  be  shortly  mentioned.  It  is  as  follows.  A  clean 
cover-glass  or  slide  is  taken  (cleaned  with  nitric  acid  and  alcohol,  or 
bichromate  of  potash  and  alcohol),  and  a  drop  of  distilled  water  placed 
upon  it.  This  is  inoculated  with  a  particle  of  a  colony  on  the  end  of  a 
platinum  needle,  and  a  scum  is  produced.  The  film  is  now  "  fixed  "  by 
slowly  drying  it  over  a  flame.  When  it  is  thus  dried,  a  drop  of  the 
selected  stain  (e.g.  gentian- 
violet)  is  placed  over  the  film 
and  allowed  to  remain  for  a  few 
seconds.  It  is  then  washed  off 
with  clean  water,  and  the  speci- 
men dried,  and  mounted  in 
Canada  balsam.  The  organisms 
will  now  appear  under  the  micro- 
scope as  violet  in  colour,  and 
FIG.  4i.— Drying  stage  for  Fixing  Films.  will  thus  be  more  clearly  seen 

than  when  unstained. 

"Double  staining"  is  adopted  when  it  is  necessary  to  stain  the 
organisms  one  colour  and  the  tissue  in  which  they  are  situated  a  contrast 
colour.  The  chief  methods  will  be  mentioned  subsequently. 

4.  Sub-culture  of  Colonies. — The  plate  method  was  introduced  by  Koch 
in  order  to  facilitate  isolation  of  species.     In  a  flask  it  is  impossible  to 
isolate   individual   species,   but   when   the    growth    is    spread    over    a 
comparatively  large  area,  such  as  a  plate,  it  is  possible  to  obtain  separate 
detached  colonies,  and  this  being  done,  the  colonies  may  be  replanted, 
by  means  of  a  platinum  wire,  in  fresh  media ;  that  is  to  say,  a  sub-culture 
may  be  made,  each  organism  cultivated  on  its  favourable  medium  and  its 
manner  of  life  closely  watched.     For  example,  a  water  may  contain  six 
species   of  bacteria.      On   the   plate   these    six    species   would    reveal 
themselves  by  their  own  peculiar  growth.     Each  would  then  be  isolated 
and  placed  in  a  separate  tube,  on  a  favourable  medium,  and  at  a  suitable 
temperature.     Thus  each  would  be  a  pure  culture  ;  i.e.,  one,  and  only  one, 
species  would  be  present  in  each  of  the  six  tubes.     By  this  simple  means 
an  organism  can  be  isolated  and  cultivated  in  the  same  sort  of  way  as  in 
floriculture.     From  day  to  day  the  habits  of  each  of  these  six  species 
may  be  observed,  and   probably   at  an  early    stage  of  their    separate 
existences   it   would   be   possible   to   determine   to  what   species   they 
belonged.     If  not,  further  microscopic  examination  could  be  made,  and, 
if  necessary,  secondary  or  tertiary  sub-cultures. 

5.  Inoculation  of  Animate. — It  may  be  necessary  to  observe  the  action 
of  supposed  pathogenic  organisms  upon  animals.     There  is  no  means  of 
testing  the  pathogenic  power  of  an  organism,  except   by  learning  by 
experiment,  whether  or  not  it  produces  disease.     As  a  matter  of  fact,  an 
immense   amount   of  bacteriological   investigation    can    be    carried    on 
without  inoculating  animals  ;   but,  strictly  speaking,  as   regards  many 
of  the   pathogenic  bacteria,  this    test   is  the    only  reliable  one.     Nor 
would  any  responsible  bacteriologist  be  justified  in  certifying  a  water 
as    healthy    for   consumption    by    a    large    community   if   he   were   in 


APPENDi 


OF  THE  ^ 

UNIVERSITY 


457 


of   any   of    the   contained 


doubt   as    to   the   disease-producing   action 
organisms. 

By  working  through  some  such  scheme  as  the  above,  it  is  possible  to 
detect  what  quantity  and  species  of  organisms,  saprophytic  or  parasitic, 
a  water  or  similar  fluid  contains.  For,  observe  what  information  has 
been  gained  by  following  out  these  five  steps  in  procedure.  We  have 
learned  the  form  (whether  bacillus,  micrococcus,  or  spirillum),  size, 
consistence,  motility,  method  of  grouping,  and  staining  reactions  of  each 
micro-organism ;  the  characters  of  its  culture,  colour,  composition, 
presence  or  absence  of  liquefaction  or  gas  formation,  its  rate  of  growth, 
odour,  or  reaction  ;  and,  lastly,  its  effect  upon  living  tissues.  Here,  then, 


FIG.  42.— Types  of  Liquefaction  of  Gelatine. 

are  ample  data  for  arriving  at  a  satisfactory  conclusion  respecting  the 
qualitative  estimation  of  the  drop  of  water  under  examination. 

As  to  the  quantitative  examination,  that  is  fulfilled  by  counting  the 
number  of  colonies  which  appear,  say  by  the  third  or  fourth  day,  upon 
the  gelatine  plates.  Each  colony  has  arisen,  it  is  assumed,  from  one 
individual,  so  that  if  the  colonies  be  counted,  though  we  do  not  thereby 
know  how  many  organisms  there  are  upon  the  plate,  it  is  known 
approximately  how  many  organisms  there  were  when  the  plate  was 
first  poured  out,  which  are  the  figures  we  require,  and  which  can  at  once 
be  multiplied  up  and  returned  as  so  many  organisms  per  drop,  or  if  the 
quantity  of  water  were  measured,  per  c.c. 

When  counting  colonies  in  a  Petri's  dish,  it  is  sufficient  to  divide  the 
circle  into  eight  equal  divisions,  and  counting  the  colonies  in  the 
average  divisions,  multiply  up  and  reduce  to  the  common  denominator  of 
1  c.c.  (or  a  Pakes'  or  Jeffer's  Counting  Disc  may  be  used).  For  example, 


458  APPENDIX 

if  the  colonies  of  the  plate  appear  to  be  distributed  uniformly,  we  count 
those  in  one  of  the  divisions.  They  reach,  we  will  suppose,  the  figure 
of  60  ;  60  x  8  =  480  micro-organisms  in  the  amount  taken  from  the  sus- 
pected water  and  added  to  the  melted  gelatine  from  which  the  plate  was 
made.  Let  us  suppose  this  amount  was  -25  c.c.  Then  the  number  of  micro- 
organisms in  the  suspected  water  is  60  x  8  =  480  x  4  =  1920  m.o.  per  c.c. 

Double  Staining1  Methods. — These  are  various,  and  are  used  when 
it  is  desired  to  stain  the  bacteria  one  colour,  and  the  matrix  or  ground 
substance  in  which  they  are  situated  another  colour.  Two  of  the 
common  methods  are  those  of  Ziehl-Neelsen  and  Gram.  They  are  as 
follows : — 

Gram's  Method. — The  primary  stain  in  this  method  is  a  solution  of 
aniline  gentian- violet  (saturated  alcoholic  solution  of  gentian-violet  30 
c.c.,  aniline  water  100  c.c.),  or  Nicolle's  carbol-gentian-violet,  which  stains 
both  ground  substance  and  bacteria  in  purple.  The  preparation  is  next 
immersed  in  the  following  solution  for  thirty  or  forty  seconds : — 

Iodine  .....  1  part 

Potassium  iodide      .  .  .  »  2  parts 

Distilled  water         .  .  .  .300  parts 

In  this  short  space  of  lime  the  iodine  solution  acts  as  a  mordant  by 
chemical  combination,  fixing  the  purple  colour  in  the  bacteria,  but  not  in 
the  ground  substance.  Hence,  if  the  preparation  be  now  (when  it  has 
assumed  a  brown  colour)  washed  in  alcohol  (methylated  spirit),  the 
ground  substance  slowly  loses  its  colour  and  becomes  clear.  But  the 
bacteria  retain  their  colour,  and  thus  stand  out  in  a  well-defined  manner. 
Cover-glass  preparations  decolorise  more  quickly  than  sections  of 
hardened  tissue,  and  they  should  only  be  left  in  the  methylated  spirit 
until  no  more  colour  comes  away.  The  preparation  may  now  be  washed 
in  water,  dried,  and  mounted  for  microscopic  examination,  or  it  may  be 
double-stained,  that  is,  immersed  in  a  contrast  stain  which  will  lightly 
colour  the  ground  substance.  Eosin  or  Bismarck  brown  are  commonly 
used  for  this  purpose.  The  former  is  applied  for  a  minute  or  two,  the 
latter  for  five  minutes,  after  which  the  specimen  is  passed  through 
methylated  spirit  (and  preferably  xylol  also)  and  mounted.  The  result 
is  that  the  bacteria  appear  in  a  dark  purple  colour  on  a  background  of 
faint  pink  or  brown.  Carbol  thionine  blue,  picro-carmine,  and  other 
stains,  are  occasionally  used  in  place  of  the  aniline  gentian-violet,  and 
there  are  other  slight  modifications  of  the  method.  The  application  of 
the  method  of  Gram  to  coverslips  and  ordinary  slide  specimens  for  the 
microscope  may  be  shortly  stated  thus  :— 

1.  Allow  two  or  three  drops  of  the  gentian-violet  stain  to  fall  upon 
slide  and  remain  in  contact  with  the  film  for  tfive  seconds.  2.  Wash  off 
the  stain  with  the  iodine  solution  applied  from  a  drop  bottle  for  five  or  six 
seconds.  The  film  should  then  be  black  or  dark  brown.  3.  Wash  off 
the  iodine  solution  with  a  mixture  of  1  part  acetone  and  2  parts  alcohol 
absolute,  but  allow  to  remain  in  contact  for  two  or  three  seconds  only. 
4.  Wash  off  with  absolute  alcohol,  applied  until  no  more  stain  comes 


APPENDIX  459 

away.  5.  Wash  in  water,  blot  off  superfluous  water,  and  set  aside  to  dry. 
If  thought  desirable,  the  preparation  may  be  counter-stained  by  the 
application  of  a  very  weak  solution  of  Ziehl-Neelsen. 

The  method  of  Gram  enables  us  to  classify  bacteria  into  two  great 
groups.  Certain  organisms  when  coloured  with  a  basic  stain  in  aniline 
or  carbolic  acid  solution,  and  afterwards  treated  with  a  special  mordant  of 
which  iodine  is  the  base,  resist  decolorisation  by  means  of  absolute 
alcohol  or  other  like  reagent.  Others,  on  the  contrary,  when  treated  in 
the  same  fashion,  readily  give  up  their  stain  and  decolorise  when  treated 
with  such  reagents.  The  Bacillus  anthracis  may  be  taken  as  a  type  of  the 
former,  the  Bacillus  typhosus  of  the  latter. 

Nicolle's  Modification  of  Gram's  Method,  used  in  the  staining  of 

diphtheria  bacillus,  consists  in  substituting  carbolic  acid  for  aniline  water, 
and  in  the  use  of  a  stronger  iodine  solution,  and  of  acetone  in  the 
decolorising  fluid.  Take  10  c.c.  saturated  alcoholic  solution  of  gentian- 
violet,  and  add  100  c.c.  of  a  1  per  cent,  solution  of  carbolic  acid.  The 
iodine  solution  consists  of  iodine  1  gramme,  potassium  iodide  2  grammes, 
and  distilled  water,  200  c.c.  Place  the  film  in  the  stain  for  five  minutes, 
then  pass  directly  into  iodine  solution  for  five  seconds,  and  decolorise  by 
passing  rapidly  through  a  mixture  of  one  volume  of  acetone  with  four 
volumes  of  absolute  alcohol.  This  removes  all  unfixed  stains  at  once.  The 
specimen  is  then  dehydrated  in  xylol,  allowed  to  dry,  and  mounted  in 
balsam. 

Ziehl-Neelsen  Method. — Here  the  primary  stain  is  a  solution  of 
carbol-fuchsin : — 

Basic  fuchsin  .  .  .  .  .  .  .1  gramme 

Absolute  alcohol  .  .  .  .  .  .     10  c.c. 

Carbolic  acid  .  .  .  .  .  .5  grammes 

Distilled  water  ,  -      -..  .  »  >      ..  .     100  c.c. 

It  is  best  to  heat  the  dye  in  a  sand  bath,  in  order  to  distribute  the 
heat  evenly.  The  various  stages  in  the  staining  process  are  as 
follows  : — («)  The  cover-glass  with  the  dried  film  upon  it  is  immersed 
in  the  hot  stain  for  one  to  three  minutes.  (6)  Remove  the  cover-glass 
from  the  carbol-fuchsin,  and,  after  washing  in  water,  place  it  in  a  capsule 
containing  a  33  per  cent,  solution  of  nitric  acid  to  decolorise  it.  Here 
its  redness  is  changed  into  a  slate-grey  colour,  (c)  Wash  in  water,  and 
alternately  in  the  acid  and  water,  until  it  is  of  a  faint  pink  colour,  (d)  Now 
place  the  cover-glass  for  a  minute  or  two  in  a  saturated  aqueous  solution 
of  methylene-blue,  which  will  counter-stain  the  decolorised  ground 
substance  blue,  (e)  Wash  in  water,  (f)  Dehydrate  by  rinsing  in 
methylated  spirit,  dry,  and  mount.  A  pure  culture  of  bacteria  will  not 
necessarily  require  the  counter-stain  (methylene-blue).  Sections  of  tissue 
may  require  twenty  to  thirty  minutes  in  a  primary  stain  (carbol-fuchsin). 

This  stain  may  be  used  for  the  bacillus  of  tubercle,  with  the 
modification  necessary  to  separate  the  bacillus  of  tubercle  from  other 
organisms  (leprosy,  etc.)  with  similar  staining  properties.  This  modifica- 
tion is  to  wash  in  absolute  alcohol,  after  the  carbol-fuchsin  stain  has  been 


460  APPENDIX 

used,  until  all  the  colour  has  entirely  disappeared.  Then  .decolorise  in 
25  per  cent,  acid  solution  for  a  few  seconds,  wash  in  water  and  alcohol 
and  acid  alternately,  and  counter-stain  as  usual.  Honsell  recommends 
acid  alcohol  (absolute  alcohol  97  per  cent.,  HC1  3  per  cent.)  for  ten 
minutes  before  counter-staining.  With  a  little  practice,  the  staining  of 
the  bacillus  of  tubercle  when  present  in  pus  or  sputum  becomes  a  simple 
and  accurate  method  of  diagnosis.  A  small  particle  of  sputum  or  pus  is 
placed  between  two  clean  cover-glasses,  and  thus  pressed  between  the 
finger  and  thumb  into  a  thin  film.  This  is  readily  dried  and  stained  as 
above.  But  washing  in  alcohol  and  acid  is  not  a  reliable  method  of 
differentiation  between  the  tubercle  bacillus  and  other  acid-fast  organisms. 
Animal  inoculation  is  the  only  reliable  test. 

Examination  Of  Moulds. — The  examination  of  hyphomycetes  or 
mould  fungi  is,  for  differentiation  purposes,  best  carried  out  on  the  Petri 
dish  itself,  where  the  construction  of  the  microscope  will  admit  of  this 
being  placed  on  the  stage. 

By  the  following  method  there  is  but  little  difficulty  in  recog- 
nising the  various  species,  and  an  excellent  demonstration  is  given  of 
the  hyphae  with  interstitial  cells,  and  germinating  conidia  of  the  Oidium 
lactis,  the  conidiophore  and  sclerotium  of  the  Pendll'mtn  glaucum,  or  the 
ramified  mycelium,  sporangia,  and  germinating  zygospores  of  the  various 
species  of  Mucor,  without  disturbance  of  the  growth.  By  means  of  a 
finely  drawn  pipette  allow  to  fall  upon  the  centre  of  the  mould  colony  a 
small  drop  of  aqueous  solution  (1  per  cent.)  of  eosin.  It  is  necessary  to 
exercise  a  little  care  in  this,  or  the  liquid  will  at  once  run  off  the  colony 
on  to  the  surrounding  medium.  Place  carefully  upon  the  centre  of  the 
drop  a  thin  cover-glass,  and  press  in  order  to  obtain  close  contact. 
Remove  the  Petri  dish  to  the  stage  of  the  microscope,  and  examine  the 
margins  of  the  growth  with  a  sixth  objective. 

If  the  construction  of  the  microscope  will  not  allow  examination  on 
the  Petri  dish,  or  if  a  permanent  specimen  is  desired,  the  following 
method  can  be  recommended  : — Detach  by  means  of  a  pair  of  fine- 
pointed  forceps  a  portion  of  the  young  growth,  holding  it  by  the  base, 
and  place  it  carefully  on  a  slide.  Place  near  it  one  drop  of  ammoniated 
alcohol,  and  bring  this  in  contact  with  the  specimen  by  means  of  a  finely 
pointed  needle.  The  absorption  of  the  alcohol  will  allow  the  subsequent 
penetration  of  the  tissues  by  the  liquids  employed.  Drop  on  to  the 
preparation  a  small  quantity  of  Fleming's  solution,  and  allow  it  to  remain 
for  four  or  five  minutes.  Wash  carefully  with  water,  cover  with  a  cover- 
glass,  and  examine. 

To  make  a  permanent  preparation,  replace  the  water  with  glycerine 
by  placing  a  drop  of  the  latter  at  one  side  of  the  cover-glass,  and  absorb 
the  water  from  the  other  by  means  of  filter-paper.  Dry  carefully  with 
filter-paper  damped  with  alcohol,  and  ring  with  paraffin. 

FLEMING'S  SOLUTION 

Chromic  acid,  1  per  cent.       .    .  .         .  .  .          15  volumes 

Osmicacid,      2        ,,      .  .  .  ,  .  4          ,, 

Glacial  acetic  acid  1  volume 


APPENDIX  461 

Flag-ella  Staining1 

Successful  staining  of  flagella  is  a  matter  of  practice,  and  of  careful 
and  exact  technique.  Whatever  the  method  of  staining  adopted,  the 
preparation  of  the  film  is  the  same,  and  too  much  care  cannot  be 
exercised  at  this  stage.  The  slides  should  in  no  case  have  been 
previously  used,  and  they  should  be  most  carefully  cleaned  in  the  manner 
described  on  p.  487.  When  taken  out  of  the  alcohol,  the  slide  should  be 
carefully  dried  and  wiped  with  a  clean  piece  of  old  cambric,  without 
handling  with  the  bare  fingers.  It  should  then  be  passed  several  times 
through  the  flame,  and  set  aside  to  cool. 

Preparation  of  the  Film. — 1.  The  cultures  for  examination  should  be 
upon  agar,  and  should  not  be  less  than  six,  or  more  than  twelve  hours 
old,  if  incubation  has  taken  place  at  37°  C.  If  incubated  at  20°,  slightly 
older  cultures  may  be  employed  (twelve  to  twenty  hours). 

2.  Transfer  from  the  young  culture  a  small  loopful  by  means  of  the 
platinum  needle,  to  a  test-tube  containing  from  30  to  40  c.c.  of  sterile 
water  at  room  temperature.     Or  the  emulsion  may  be  made  in  a  capsule 
with  a  few  c.c.  of  distilled  water.     Hold  the  loop  in  the  water  for  a 
few  moments  without  shaking,  until  the  water  shows  a  slight  turbidity. 
Do  not  shake  or  handle  the  tube  roughly.     Incubate  the  emulsion  for 
five  hours  at  37°  C.  or  for  twelve  to  twenty-four  hours  at  20°  C. 

3.  With  a  finely  looped   pipette,  take  up  a  small  quantity  of  the 
surface  water  from  the  inoculated  tube,  and  distribute  it  in  small  droplets, 
upon  the  slide. 

4.  Place  aside  to  dry,  carefully  covered  from  chance  of  dust.     When 
dry,   the   staining  can    be   proceeded  with,   according   to   the   method 
adopted.     Do  not  fix  the  films  in  the  flame ;  the  flagella  are  apt  to  be 
injured  thereby,  and  it  will  be  found  that  the  subsequent  manipulations 
will  cause  the  organisms  to  adhere  sufficiently  to  the  slide. 

Staining  the  Film 

The  three  ordinary  methods  practised  in  this  country  are  : — 

(1)  PitfieWs  Method  (Muir's  modification). 
The  following  solutions  are  required : — 

A.  The  Mordant. 

Tannic  acid,  10  per  cent,  aqueous  solution  .  .  10  c.c. 

Corrosive  sublimate,  saturated  aqueous  solution  .  .  5  c.c. 

Alum,  saturated  aqueous  solution  .            .  .  .  5  c.c. 

Carbol-fuchsin  (Ziehl)          .            .            .  .  5  c.c. 

The  above  must  be  thoroughly  mixed  and  the  precipitate  which  forms  must  be 
allowed  to  deposit.  The  clear  supernatant  fluid  is  then  drawn  off  with  a  pipette  and 
placed  in  a  clean  dropping  bottle.  The  mordant  will  remain  good  for  one  or  two 
weeks,  but  not  longer.  It  should  be  centrifugalised  before  use. 

B.  The  Stain. 

Alum,  saturated  aqueous  solution  .  .  .       25  c.c. 

Gentian- violet,  saturated  alcoholic  solution  .  .        5  c.c. 

Filter  twice.     The  stain  must  be  freshly  prepared. 

The  film  is  prepared  as  described  above.     The  mordant  is  then  dropped  on  to 


462  APPENDIX 

the  slide  and  heated  gently  over  the  flame  until  the  steam  begins  to  rise.  Allow  to 
steam  for  from  one  to  two  minutes  ;  wash  well  in  running  water  and  dry  carefully. 
When  thoroughly  dry,  apply  a  sufficient  quantity  of  the  stain,  and  heat  as  before, 
allowing  to  steam  for  two  minutes.  Wash  in  distilled  water,  dry,  and  examine. 

(2)  Van  Ermengem's  Method. 

Three  solutions  are  required  in  this  method : — 

A.  Fixing  Solution. 

Osmic  acid,  2  per  cent,  aqueous  solution  .  .  .       10  c.c.  * 

Tannin,  20  per  cent,  solution  .  .  .  .       20  c.c. 

To  each  100  c.c.  of  this  mixture  add  4  to  5  drops  of  glacial  acetic  acid.  The 
colour  of  this  solution  should  be  violet,  and  the  solution  should  be  filtered  before  use. 

B.  Sensitising  Solution. 

Nitrate  of  silver         .  .  .  .      0  *5  aqueous  solution 

This  solution  should  be  kept  in  the  dark,  and  filtered  before  use. 

C.  Reducing  Solution. 

Gallic  acid      ......  5  grammes 

Tannin 3 

Fused  acetate  of  soda  (or  potassium)           .            .  10         ,, 

Distilled  water           .....  350         „ 

(a)  Cover  the  film  with  solution  "A,"  and  allow  to  act  for  five  minutes  at  37°  C., 
or  one  hour  at  room  temperature.  Or  heat  gently  until  steam  rises,  and  allow  the 
staining  fluid  to  act  for  five  minutes. 

(6)  Wash  well  with  distilled  water,  then  in  absolute  alcohol,  and  then  again  in 
distilled  water. 

(c)  Treat  with  solution  *•  B,"  and  allow  it  to  act  for  thirty  seconds,  keeping  the 
fluid  in  movement  on  the  slide. 

(d)  Allow  the  fluid  to  run  off  the  slide,  and  without  washing  treat  with  •*  C  "  for 
thirty  seconds  in  the  same  manner. 

(e)  Allow  fluid  to  run  off,  and  again  treat  with  **  B  "  until  the  preparation  begins 
to  turn  black. 

(/)  Wash  in  distilled  water,  mount  in  water,  and  examine  under  the  microscope. 
The  method  is  not  wholly  satisfactory. 

A  simple  method  is  as  follows  : — 

(3)  Night-Blue  Method  (M'Crorie). 

Place  2  or  3  drops  of  the  emulsion  on  an  absolutely  clean  slide,  and  dry  at  room 
temperature.  It  is  not  necessary  or  desirable  to  fix  by  heat  The  stain  is  made  by 
mixing  10  c.c.  of  night-blue,  saturated  alcoholic  solution,  10  c.c.  of  a  saturated 
aqueous  solution  of  potash  alum,  and  10  c.c.  of  a  10  per  cent,  aqueous  solution  of 
tannin.  The  stain  must  be  filtered  before  use.  The  slides,  as  prepared  above,  are 
stained  with  this  for  two  minutes  in  the  hot  incubator,  and  then  washed  gently  in 
running  water.  It  may  be  found  best  to  change  the  blue  stain  several  times  during 
the  two  minutes.  A  counter-stain  may  be  used  if  desired,  and  one  of  the  best  is 
aniline  gentian-violet.  This  should  be  applied  for  about  a  minute,  after  which  a 
cover-glass  may  be  fixed  over  the  film  with  Canada  balsam.  In  such  a  preparation 
the  bacilli  will  be  stained  violet  and  the  flagella  blue.  Better  results  may  sometimes 
be  obtained  by  staining  deeply  with  the  blue  (ten  minutes),  and  then  decolorising  to 
the  necessary  extent  in  dilute  methylated  spirit 

The  Staining1  of  Spores 

The  following  are  the  methods  commonly  adopted  : — 

(1)  Roller's  Method. 

(a)  Prepare  the  film  as  usual,  fix  and  dry,  observing  the  precautions  taken  in 
preparing  milk  specimens. 


APPENDIX  463 

(l>)  Treat  with  alcohol  for  two  minutes,  and  then  with  chloroform  for  two  minutes  ; 
wash  in  water. 

(c)  Treat  with  chromic  acid,  5  per  cent,  aqueous  solution,  for  from  one  to  two 
minutes  ;  wash  and  dry. 

(d)  Pour  on  freshly  filtered  carbol-fuchsin  and  warm  gently  till  it  steams  ;  allow 
it  to  act  for  ten  minutes  and  wash  off  with  water. 

(e)  Decolorise  with  sulphuric  acid  (5  per  cent. )  and  water  alternately,  to  remove 
the  carbol-fuchsin  from  the  bacilli  but  not  the  spores. 

(/)  Dry  and  counter-stain  with  Loffler's  blue  until  the  film  is  of  a  faint  bluish 
tint.  Wash  off  stain,  dry  and  examine.  The  spores  will  be  stained  red  and  the 
bacilli  blue. 

(2)  Ziehl-Neelsen  Method. 

(a)  Stain  the  film  as  for  tubercle  bacilli. 

(/>)  Decolorise  with  1  per  cent,  aqueous  solution  of  sulphuric  acid,  or  alcohol  2 
parts,  acetic  acid  1  per  cent.,  1  part. 

(c)  Counter-stain  with  Loffler's  blue. 

(d)  Wash,  dry,  and  examine. 

(3)  Abbott's  Method. 

Prepare  films  in  usual  way,  and  stain  with  Loffler's  alkaline  methylene-blue, 
heating  gently  till  steam  rises  (5  minutes).  Then  wash  in  water  and  decolorise  with 
nitric  acid,  2  per  cent,  alcoholic  (80  per  cent.)  solution,  washing  again  in  water. 
Counter-stain  with  eosin,  1  per  cent,  aqueous  solution.  Wash,  dry,  and  mount. 
The  spores  are  blue  and  the  bacilli  red. 

Bacteriological  Diagnosis. — The  following  points  must  be  ascer- 
tained in  order  to  identify  any  particular  micro-organism  : — 

(1)  Its  morphology  :  shape,  size,  etc.  (bacillus,  coccus,  spirillum,  etc.)  ; 
the  presence  or  absence  of  involution  forms ;  motility,  by  the  unstained 
cover-glass  preparation  ("hanging  drop");    note  presence  of  flagella ; 
presence  of  spores,  their  appearance  and  position.     Staining  reaction ; 
whether  or  not  the  organism  stains  by  Gram's  method. 

(2)  Cultural  Characters. — The  character  of  the  growth  upon  various 
media   (gelatine,  agar,  milk,  potato,  blood    serum,  broth,   and    special 
media) ;  the  presence  or  absence  of  liquefaction  in  the  gelatine  culture ; 
its  power  of  producing  pigment,  acid,  gas,  indol,  ferments,  phenol,  etc. 

(3)  Biology  :  whether  it  is  aerobic  or  anaerobic  ;  its  powers  of  resist- 
ance to  external  agencies  ;  agglutination  reaction,  etc. ;  pathogenesis,  its 
effect  upon  animal  tissues  and  the  course  of  the  disease  produced ;  its 
toxins,  etc. 

BACTERIOLOGICAL  EXAMINATION   OF   WATER 

Collection  of  Samples, — Water  from  streams  or  wells  should  be 
collected  in  glass  bottles  or  flasks  closed  with  glass  stoppers  previously 
sterilised  (at  150°  C.  for  three  hours),  or  washed  out  with  pure  sulphuric 
acid.  When  the  latter  method  is  adopted,  the  bottle  should  be  well  rinsed 
with  the  water  which  is  to  be  examined  before  the  sample  is  taken.  In 
taking  the  sample,  the  bottle  should  be  held  below  the  surface  before  the 
stopper  is  removed,  in  order  to  obtain  a  sample  of  the  main  body  of  water 
and  not  the  surface  water  only.  If  it  is  an  ordinary  water  supply 
through  pipes  or  from  a  cistern,  the  tap  should  be  turned  on  and  the 
water  allowed  to  run  for  a  few  minutes  before  taking  the  sample  :  and 


464 


APPENDIX 


the  same  principle  applies  to  a  well  not  in  regular  use.  Such  a  well 
should  be  pumped  for  some  time  before  taking  the  sample.  For  obtain- 
ing samples  from  a  considerable  depth  Miquel's  apparatus  may  be  used, 
or,  if  that  is  not  available,  a  weighted  bottle. 

After  collection,  the  bottle  should  be  at  once  stoppered,  labelled,  and 
packed  in  ice  and  sawdust  for  transport  to  the  laboratory,  or  placed  in 
one  of  the  various  ice  cases  now  in  use  (Delepine's  or  Pakes').  Below 
5°  C.,  organisms  do  not  multiply  in  water,  and  therefore  it  is  important 
to  keep  samples  previous  to  examination  at  a  low  temperature.  In  all 
cases  where  it  is  possible,  the  water  should  be  examined  at  once  after 
collection. 

Physical  Examination. — The  temperature  and  reaction  of  the  water 
should  first  be  tested,  and  an  examination  made  of  any  deposit  or 
suspended  matter.  Bubbles  of  gas,  if  present,  should  be  noted.  The 
colour,  character,  and  amount  of  particulate  matter  in  suspension  or 
sediment  should  be  observed  and  noted  ;  turbidity,  odour,  flavour  and 
taste,  peatiness,  etc.,  should  all  be  noted.  A  record  of  the  quantity  of 
the  sample,  its  source,  and  the  date  and  time  of  its  collection  is  also 
important.  A  microscopical  examination  of  the  matter  obtained  by 
filtration  followed  by  centrifugalisation  may  also  yield  important  facts. 

Bacteriological  Examination. — This  divides  itself  naturally  into  two 
divisions — (a)  a  quantitative  examination,  and  (V)  a  qualitative  examina- 
tion.* 

0)  Quantitative  Examination 

The  sample  should  be  gently  mixed,  and  plate  cultivations  made. 
Take  five  tubes  of  10-15  c.c.  of  gelatine  and  five  Petri  dishes,  and  melt 
the  medium  of  the  former  in  a  water  bath.  The  gelatine  should  be  well 
liquefied,  but  not  overheated.  The  Petri  dishes  should  be  of  even 
surface,  equal  size,  and  properly  sterilised.  Take  a  1  c.c.  sterilised 


r%.  ^^n 


FIG.  43.— Levelling  Apparatus  for  Koch's  Plate. 


FIG.  44.— Moist  Chamber  for  Koch's 
Plate. 


pipette  accurately  calibrated,  and  pass  it  into  the  bottle,  removing  the 
necessary  quantities  of  water.  As  a  rule,  0*5  c.c.,  0*2  c.c.,  O2  c.c., 
O'l  c.c.,  and  0*1  c.c.  are  suitable  quantities  for  each  of  the  five  plates. 
Add  these  quantities  to  the  five  tubes  of  liquefied  gelatine,  and  gently 

*  An  admirable  illustration  of  how  to  examine  a  water  is  furnished  in  the  Report 
of  Medical  Officer  to  Local  Government  Board,  1901-2,  pp.  494-547  (Houston). 


APPENDIX 


46J 


mix  and  pour  into  the  Petri  dishes.  Allow  the  gelatine  to  set ;  and 
incubate  at  22°  C.  for  as  long  as  possible  before  complete  liquefaction 
occurs.  Count  the  colonies  which  appear  after  forty-eight  hours  incuba- 
tion (agar),  take  the  average  at  the  period  of  maximum  growth  (gelatine 
4-5  days),  multiply  up  according  to  the  fraction  of  a  c.c.  which  has  been 
used,  and  return  as  so  many  organisms  per  cubic  centimetre,  stating 
medium,  period  and  temperature  of  incubation,  etc.  It  is  advisable  that 
each  quantity  of  water  from  which  the  fractional  part  is  added  to  the 
gelatine  should  be  taken  up  separately,  and  not  that  1  c.c.  of  water  should 
be  taken  up  and  the  fractional  amounts,  say  of  0-5,  0'2,  and  O'l  c.c.,  be 
added  to  the  gelatine.  If  Koch's  plates  are  used  they  should  be  allowed 


FIG.  45.— Wolfhiigel's  Counter. 

to  set  on  the  levelling  apparatus,  then  placed  in  the  moist  chamber  for 
incubation  at  22°  C.,  and  the  colonies  counted  by  means  of  Wolfhiigel's 
counter  (see  Figs.  43,  44,  and  45). 

(b)  Qualitative  Examination 

At  the  time  of  making  the  gelatine  plates  for  quantitative  examina- 
tion, several  agar,  and  litmus-lactose  agar,  plates  may  be  made  for  quali- 
tative purposes.  The  plates  must  be  poured  immediately  after  inoculation 
of  liquefied  agar  with  small  quantities  of  the  water,  as  below  40°  C.  the 
agar  will  resolidify.  When  poured,  the  agar  plates  should  be  placed  on 
cold  *stone  or  metal,  and  then  incubated  at  blood-heat.  On  the  second 
or  third  day  colonies  will  have  appeared,  and  these  should  be  studied 
and  sub-cultured  (as  pure  cultures)  on  suitable  media. 

Valuable  facts  as  to  the  quality  of  the  water  may  also  be  obtained 
from  an  examination  of  the  five  gelatine  plates,  particularly  in  respect 
of  the  liquefying  organisms,  which  should  be  counted  as  carefully  as  any 
other  colonies,  and  noted  separately  as  well  as  in  the  total  number  of 
colonies  present.  But  in  addition  to  the  facts  obtained  from  gelatine 
and  agar  plates,  other  methods  must  be  adopted  in  order  to  obtain 
information  respecting  the  quality  of  the  water. 

Take  a  sterilised  Berkefeld  porcelain  filter,  and  pump  or  aspirate 
through  it  1000-2000  c.c.  of  the  water  under  examination,  and  with  a 

2  G 


466 


APPENDIX 


sterilised  brush,  transfer  the  participate  matter  which  has  collected  on 
the  candle  into  10  c.c.  of  sterile  water  or  broth.  This  is  now  a  concen- 
tration or  emulsion  of  the  organismal  content  of  the 
litre  of  water,  and  may  be  used  for  examination  for 
special  organisms. 

(a)  B.  enteritidis  sporogrenes.— Place  0-5  or 

1  c.c.  of  the  concentrated  water  in  each  of  three 
tubes  of  10-15  c.c.  of  fresh  sterilised  milk.  It  is 
important  to  use  fresh  milk,  recently  boiled,  and 
cooled  down  before  inoculation.  After  inoculation 
with  the  water  to  be  examined,  put  the  three  tubes 
into  the  water  bath  for  fifteen  minutes  at  80°  C.,  and 
after  allowing  them  to  cool,  place  them  in  a  Buchner's 
tube  or  cylinder  containing  freshly-prepared  pyro- 
gallic  solution  (pyrogallic  acid,  120  grains,  strong 
liquor  potassae,  10  c.c.).  Accurately  seal  up  the 
Buchner,  and  place  it,  containing  the  tubes,  in  the 
incubator  at  37°  C.  The  next  day,  or  in  forty-eight 
hours,  examine  for  B.  enteritidis  sporogenes.  If  that 
organism  is  present,  the  following  characteristic 
appearances — the  enteritidis  change — will  be  ap- 
parent (Klein).  The  cream  of  the 
milk  will  be  torn  and  altogether 
dissociated  by  the  development  of 


Fia.  46.— Filter  in  posi- 
tion for  filter-brushing 
method. 


gas,  so  that  the  surface  of  the 
medium  becomes  covered  with 
stringy  white  masses  of  coagulated 
casein,  enclosing  a  number  of  gas 
bubbles.  The  main  portion  of  the  tube  formerly  occu- 
pied by  the  milk  will  contain  a  colourless  thin  watery 
whey,  with  a  few  lumps  of  casein  adhering  here  and 
there  to  the  sides  of  the  tube  (see  Plate  21,  p.  307).  If 
the  tube  be  opened,  there  will  be  found  to  be  an  odour  of 
butyric  acid  and  an  acid  reaction.  If  some  of  the  con- 
tents of  the  tube  are  stained,  as  slide  preparations,  the 
bacilli  will  be  seen. 

(b)  B.  coli  communis  (p.  46).— Take  from  0-1 

to   0*5    of    the    concentrated    or    sample    water,    and 

add   to   tubes    of   phenolated    gelatine    ('05  per  cent. 

phenol),    or    litmus-lactose    agar,    and    make    plates. 

Colonies  developing  in  these  plates  (red  in  latter  medium)  should  be 

suspected  of  being  B.  coli  communis,  and  tested  accordingly  ; 

Or,  inoculate  from  the  concentrated  or  sample  water,  three  tubes  of 
Parietti's  broth,*  and  incubate  at  37°  C.,  and  those  tubes  which  show 

*  Parietti's  Formula  consists  of — phenol,  5  grams  ;  hydrochloric  acid,  4  grams  ; 
distilled  water,  100  c.c.  To  10  c.c.  of  broth,  0  '1-0  '3  c.c.  of  this  solution  is  added.  The 
tube  is  then  incubated  in  order  to  test  its  sterility.  If  it  be  sterile,  a  few  drops  of  the 
suspected  water  are  added,  and  the  tube  reincubated  at  37°  C.  for  twenty-four  hours. 
If  the  water  contains  the  B.  typhosus  or  B,  coli,  the  tube  will  show  a  turbid  growth. 


FIG.  47.— Buchner 
Tube. 


PLATE  31. 


APPARATUS  FOR  FILTERING  WATER  TO  FACILITATE  ITS  BACTERIOLOGICAL  EXAMINATION. 
(The  filter-brushing  method). 


[To  face  page 


APPENDIX  467 

growth  in  one  to  three  days  should  be  plated  out  on  ordinary  or  pheno- 
lated  gelatine  and  colonies  of  B.  coli  examined  for.  Some  authorities 
recommend  incubating  Parietti's  tubes  at  42°  C.,  a  temperature  favour- 
able to  B.  coli  but  unfavourable  to  ordinary  water  organisms  ; 

Or,  tubes  of  glucose-formate  bouillon  (meat  infusion,  1  per  cent, 
peptone,  0-5  per  cent,  salt,  2  per  cent,  glucose,  and  0'4  sodium  formate) 
may  be  inoculated  with  Ol  to  0*5  c.c.  of  the  water,  and  incubated  in 
Buchner's  tube  at  42°  C.,  and  the  tubes  which  show  turbidity  in  24  hours 
may  be  plated  out  on  gelatine  or  glucose-litmus  agar  and  B.  coli,  if 
present,  thus  isolated  (Pakes'  method) ; 

Or,  inoculate  tubes  of  M'Conkey's  medium,  bile-salt-glucose  peptone, 
with  1*5,  or  10  c.c.  of  the  water.  "When  this  test  yields  negative  results, 
the  absence  of  B.  coli  and  of  glucose  fermenting  coli-like  microbes  may 
be  accepted  without  reserve  "  (Houston)  (see  p.  484)  ; 

Or,  place  1-20  c.c.  of  the  water,  or  '01-'5  of  the  suspension,  into  tubes 
of  bile-salt  solution,  and  incubate  at  37°  C.  aerobically  for  twenty-four 
hours,  or  at  42°  C. anaerobically  (in  Buchner's  tubes)  for  twenty-four  hours; 
and  if,  after  this  period,  there  is  (a)  presence  of  growth,  (6)  formation 
of  acid,  or  (c)  formation  of  gas,  plate  out  on  gelatine  agar  or  bile-salt- 
lactose-peptone  agar,  and  sub-culture  coli-like  colonies  on  suitable  media  ; 

Or,  incubate  at  38°  C.  1  c.c.  of  the  water  in  a  Smith's  fermentation 
tube  with  glucose  broth.  If  after  twelve  hours'  growth  gas  collects,  it 
may  be  B.  coli,  and  the  species  must  be  further  tested.  If  there  is  no 
gas  there  is  probably  no  B.  coli. 

In  the  examination  for  B.  coli,  the  important  media  for  sub-culturing 
are  as  follows  :  gelatine  shake  cultures  *  (for  gas  production  and  lique- 
faction), glucose  gelatine  or  glucose  agar  (for  gas  production),  milk  or 
litmus  milk  (for  acidity  and  coagulation),  peptone  water  (for  indol),f  and 
potato.  Eisner  medium,  J  neutral-red  agar,  lactose  and  maltose  media  may 
also  be  used. 

*"  Shake  Cultures."" — To  10  c.c.  of  melted  gelatine,  a  small  quantity  of  the 
suspected  organism  is  added.  The  test-tube  is  then  shaken  and  incubated  at  22°  C. 
In  this  medium  the  B.  coli  have  opportunity  for  gas  production. 

•[The  Indol  Reaction. — Indol  and  skatol  are  amongst  the  final  products  of 
digestion  in  the  lower  intestine.  They  are  formed  by  the  growth,  or  fermentation 
set  up  by  the  growth,  of  certain  organisms.  Indol  may  be  recognised  on  account  of 
the  fact  that  with  nitrous  acid  it  produces  a  dull  red  colour.  The  method  of  testing 
is  as  follows.  The  suspected  organism  is  grown  in  pure  culture  in  broth  or  peptone 
water  (or  Dunham's  solution),  and  incubated  for  forty-eight  hours  at  37°  C.  Two  c.c. 
of  a  '01  per  cent,  solution  of  potassium  nitrite  is  added  to  the  test-tube  of  broth 
culture.  Now  1  c.c.  of  concentrated  sulphuric  acid  (unless  quite  pure,  hydrochloric 
should  be  used)  is  run  down  the  side  of  the  tube.  A  pale  pink  to  dull  red  colour 
appears  almost  at  once,  or  in  a  few  minutes,  and  may  be  accentuated  by  placing  the 
culture  in  the  blood-heat  incubator  for  half  an  hour.  The  presence  of  much  dextrose 
(derived  from  the  meat  of  the  broth)  inhibits  the  reaction.  B.  typhosus  does  not 
produce  indol,  and  therefore  does  not  react  to  the  test ;  B.  coli  and  the  bacillus  of 
Asiatic  cholera  do  produce  indol,  and  react  accordingly. 

J  Eisner's  Medium.  — This  special  potassium-iodide-potato  gelatine  medium  is  used 
for  the  examination  of  typhoid  excreta.  It  is  made  as  follows  :  500  grams  of  potato 
gratings  are  added  to  1000  c.c.  of  water;  stand  in  ice-chest  for  twelve  hours,  and 
filter  through  muslin;  add  150  grams  of  gelatine ;  sterilise  and  add  enough  deci- 


468  APPENDIX 

Differential  Diagnosis  of  B.  coli. — The  general  characters  of  B.  coli 
will  be  found  in  the  text  of  the  present  volume,  but  it  may  be  stated 
for  diagnostic  purposes  that  most  reliance  should  be  placed  upon  the 
following  characters.  (But  all  characters  must  be  taken  into  considera- 
tion in  forming  an  opinion.)  B.  coli  produces  a  characteristic  growth 
on  gelatine  plate,  smooth,  milk-white  colonies ;  produces  gas  in 
lactose,  saccharose  and  glucose  media ;  is  motile,  non-liquefying  (up 
to  the  14th  day)  and  does  not  stain  by  Gram;  produces  acid 
curdling  of  milk  within  four  days  at  37°  C.  The  production  of  a 
yellowish-green  fluorescence  in  neutral-red  agar  shake  culture  and 
the  production  of  indol  in  peptone  water  or  broth  (but  without 
pellicle)  are  further  tests  relied  upon  by  some.  The  Lawrence  method 
(State  Board  of  Massachusetts)  of  testing  for  B.  coli  includes  the  follow- 
ing seven  tests  :  (a)  characteristic  appearance  on  agar  streak,  (//)  growth 
on  litmus-lactose  agar,  (c)  gas  production  in  dextrose  broth,  (d)  coagula- 
tion of  milk,  (e)  production  of  nitrites  in  nitrate  broth,  (/')  production 
of  indol  in  Dunham's  solution,  and  (g)  non-liquefaction  of  gelatine.* 

B.  lactis  aerogenes  is  similar  to  B.  coli,  but  coagulates  milk  much  more 
slowly  and  is  non-motile. 

B.  Gaertner  and  its  allies  ferment  glucose  but  riot  lactose  in  litmus 
milk ;  cultures  are  generally  acid  at  first,  and  subsequently  alkaline,  and 
there  is  no  coagulation. 

B.  typhosus  produces  no  gas  in  any  media,  does  not  coagulate  milk, 
stains  by  Gram,  and  serum  diagnosis  is  also  practicable. 

Proteus  group  are  similar  to  B.  coli,  except  that  they  liquefy  gelatine 
and  are  slow  in  curdling  milk. 

(c)  B.  typhOSUS  may  be  examined  for  by  adopting  exactly  the  same 
methods   as  for   B.  coli.     Its    detection   in,  and   isolation    from,   water 
supplies  is  so  difficult  as  to  be  well-nigh  impossible.     The  condition  of 
a  water  is,  however,  ascertainable  short  of  an  absolute  test  for  B.  typhosus, 
valuable  though  that  would  be. 

(d)  For  the  detection  of  the  cholera  spirillum,  add  10  c.c.  of  peptone 
solution  (10  per  cent,  peptone,  20  per  cent,  gelatine,  and  5  per  cent, 
salt)  to  90  c.c.  of  the  water  to  be  tested.     Incubate  at  37°  C.     After 
twelve   to   twenty-four   hours   incubation,    examine   loopfuls    from    the 
surface  pellicle  for    spirilla ;   or  plate  out  loopfuls  of  the  pellicle   on 
gelatine  and  agar ;  or  test  for  cholera  red  reaction  and  Pfeiffer's  reaction 
and  agglutination  test ;    or  culture    emulsion   from   Berkefeld    filter  in 
peptone  water,  and  then   plate  out  on  gelatine  and  agar  from  tubes 
showing  pellicle. 

(e)  For  the  detection  of  StreptOCOCCi,  plate  out  the  emulsion  obtained 
from  the  Berkefeld  filter  on  agar,  and  incubate  at  37°  C.     After  forty- 
normal  caustic  soda  until  only  faintly  acid ;  add  white  of  egg ;  sterilise  and  filter. 
Before  use  add  a  gram  of  potassium  iodide  to  every  100  c.c.     Filter,  and  sterilise  a 
100°  C.  for  twenty  minutes  on  three  successive  days.      Upon  this  acid  medium 
common  water  ibacteria  will  not  grow,  but  B.  typhosus  and  B.  coli  flourish — the 
former  like  "small  clear  droplets,"  the  latter  as  dark  brown  globular  masses. 

*  Report  of  State  Board  of  Health,  Massachusetts,  1901,  p.  400  ;  ibid.,  1902,  pp.  262 
and  280. 


APPENDIX  469 

eight  hours  examine  the  plate  with  a  lens,  and  pick  out  the  minute 
colonies,  streptococci,  and  sub-culture  in  broth,  and  incubate  at  37°  C. 
Stain  by  Gram's  method,  and,  if  necessary,  further  sub-culture. 

(/)  Sewage  Organisms  and  the  organisms  indicative  of  surface 
pollution  should  also  be  examined  for.  If  they  be  present  in  the  water, 
it  may  be  taken  as  proved  that  such  water  has  been  recently  polluted, 
and  should  be  condemned.  Crude  sewage  generally  contains  in  1  c.c. : 
(a)  1  to  10  million  bacteria;  (6)  100,000  B.  coli  (or  closely  allied  forms)  ; 
(c)  100  spores  of  B.  enterilidis  spor o genes ;  and  (d~)  1000  streptococci 
(Houston).  Further,  so  minute  a  quantity  as  y^Vzy  of  a  c.c.  of  crude 
sewage  is  usually  sufficient  to  produce  "gas"  in  a  gelatine  "shake" 
culture  in  twenty-four  hours  at  20°  C.,  and  the  inoculation  of  animals 
with  crude  sewage  always  leads  to  a  local  reaction  and  not  uncommonly 
results  in  death.  These  three  organisms,  B.  coli,  B.  enteritidis  sporogenes, 
and  streptococci  have  been  termed  the  "  microbes  of  indication."  These 
bacteria  are  wholly,  or  relatively,  absent  from  pure  water,  and  their 
presence,  at  all  events  in  considerable  numbers,  must  be  taken  as 
indicating  recent  animal  pollution*  B.  coli  is  a  most  accurate  measure  of 
intestinal  pollution,  and  far  greater  information  as  regards  the  sewage 
pollution  of  water  can  be  gathered  by  its  estimation,  than  by  simply 
counting  the  total  number  of  organisms  present  in  water.  It  is  an 
intestinal  parasite,  and  tends  to  perish  in  other  media. f  When  it  is 
present  in  a  small  stream,  contamination  from  houses  can  be  traced.  £ 

Thresh  has  suggested  the  following  scheme  of  examination  of  a 
water  as  one  furnishing  the  minimum  amount  of  information  which  will 
enable  anyone  to  say  positively  that  a  water  is  fsecally  contaminated : — 
(1)  The  detection  of  the  presence  of  organisms  of  intestinal  type ;  (2) 
the  isolation  and  identification  of  B.  coli;  and  (3)  the  detection  of  the 
presence  of  spores  of  B.  enteritidis  sporogenes.  The  process  he  recom- 
mends is  as  follows :— (a)  Make  bile-salt  broth  cultures  with  1,  5,  10, 
and  20  c.c.  of  the  water  to  be  examined.  After  twenty-four  hours  the 
tube  containing  the  smallest  quantity  of  water  showing  acid  and  gas 
formation  is  selected  for  further  examination.  If  after  forty-eight  hours 
there  is  no  such  reaction,  no  further  examination  is  made.  (6)  Two  or 
three  loopfuls  of  the  culture  are  added  to  10  c.c.  of  sterilised  water,  and 
a  loopful  of  the  solution  is  spread  over  a  plate  of  bile-salt-lactose- 
peptone  agar  containing  neutral  red  and  made  faintly  alkaline  to  litmus. 
This  plate  is  incubated  for  twenty-fours  at  37°  C.,  and  the  colonies  pro- 
duced carefully  examined.  As  the  B.  coli  communis  ferments  lactose 
with  the  production  of  acid,  any  colonies  of  this  organism  will  be  of  a 
red  colour  and  be  surrounded  by  a  haze,  formed  by  the  precipitation  of 
the  bile  acids.  As"  this  haze  may  not  be  apparent  at  the  end  of  twenty- 
four  hours,  types  of  all  the  red  colonies  are  taken  for  the  further 
examination,  (c)  Each  colony  so  selected  is  used  to  inoculate  a  tube  of 
lactose-peptone-bile-salt-litmus  solution,  and  after  twenty-four  hours' 
incubation,  if  acid  and  gas  is  produced,  the  growth  is  examined  micro- 

*  Second  Report  of  Royal  Commission  on  Sewage  Disposal,  1902,  pp.  26  and  27. 
t  Ibid.,  p.  99.  £  /&&,  p.  109. 


470  APPENDIX 

scopically  to  ascertain  if  the  bacillus  is  motile  or  not  (or  spore-bearing), 
and  it  is  then  treated  by  Gram's  method  to  ascertain  whether  it  retains 
the  stain.  The  results  being  recorded,  (d)  The  turbid  fluid  is  used  to 
inoculate — Litmus  milk,  for  acid  and  clotting  ;  glucose- neutral-red  agar, 
for  gas  and  fluoresence ;  gelatin  (stab  or  streak),  for  absence  of  liquefac- 
tion ;  and  peptone  solution,  for  indol.  If  the  B.  coli  is  present,  all  the 
reactions  indicated,  with  the  possible  exception  of  the  production  of 
fluorescence,  will  be  produced. by  one  or  more  of  the  colonies  selected. 
The  water  is  tested  for  the  presence  of  B.  enteritidis  sporogenes  in  the 
ordinary  way.  It  should  be  added  that  such  an  examination  as  the 
above  fails  to  obtain  information  upon  the  general  constitution  or  the 
bacteriological  flora  of  a  water  which  is  obtained  by  the  additional 
means  of  the  gelatine  plate  method,  and  whilst  of  value  for  rapid  use, 
should  not  be  substituted  for  the  systematic  study  of  a  water. 


REPORT  OF  THE  COMMITTEE  APPOINTED  BY  THE  ROYAL  INSTI- 
TUTE OF  PUBLIC  HEALTH  TO  CONSIDER  THE  STANDARDISA- 
TION OF  METHODS  FOR  THE  BACTERIOSCOPIC  EXAMINATION 
OF  WATER,  1904. 

All  the  members  of  the  committee  are  in  agreement  that  the  minimal  number  of 
procedures  should  be : 

(a)  Enumeration  of  the  bacteria    present  on  a  medium  incubated  at  room 

temperature  (18°-22°  C.). 
(6)  Search  for  B.  coli,  and  identification  and  enumeration  of  this  organism  if 

present. 

The  committee  regard  these  procedures  as  an  irreducible  minimum  in  the 
bacterioscopic  analysis  of  water.  The  majority  of  the  committee  recommend  in 
addition : 

(c)  Enumeration  of  the  bacteria  present  on  a  medium  incubated  at  blood-heat 

(36°-38°C.). 

(d )  Search  for  and  enumeration  of  streptococci. 

The  committee  do  not  think  it  necessary  as  a  routine  measure  to  search  for  the 
B.  enteritidis  sporogenes,  but  are  agreed  that  in  special  or  exceptional  instances  it 
may  be  advisable  to  look  for  this  organism. 

The  Collection  of  the  Sample. — No  special  precautions  beyond  those  generally 
recognised  are  suggested  for  taking  the  sample.  The  samples  should  be  collected 
in  sterile  stoppered  glass  bottles  having  a  minimal  capacity  of  60  c.c.  In  special 
instances  it  may  be  desirable  to  have  much  larger  quantities. 

Unless  examined  within  three  hours  of  collection  the  sample  must  be  ice-packed. 

(The  committee  recognise  that  under  all  circumstances  the  sooner  the  water  is 
examined  after  collection  the  more  reliable  are  the  results  obtained.) 

Media  to  be  employed  for  Enumeration. — The  choice  of  medium  lies  between 
distilled-water  gelatin,  nutrient  gelatin,  distilled- water  agar,  gelatin  agar,  and 
nutrient  agar.  The  reaction  of  the  medium  is  of  importance. 

For  enumeration  at  room  temperature,  any  of  these  media  may  be  employed  ; 
but  for  enumeration  at  blood-heat,  an  agar  or  gelatin  agar  must  be  used. 

The  Americans  seem  to  be  using  an  agar  medium  only,  and  although  on  the 
ground  of  simplicity  it  might  be  desirable  to  use  a  single  medium  for  enumeration 
under  all  circumstances— e.g.  a  distilled-water  agar— it  is  felt  by  the  committee 
that  gelatin  media  frequently  give  indications  of  value  that  are  lacking  with  agar — 
vi/.,  liquefaction  of  the  medium  by  many  organisms  and  the  more  characteristic 
appearance  of  the  colonies  in  it ;  gelatin  is  therefore  recommended. 

Since  with  a  polluted  water  (detection  of  pollution  being  the  ultimate  aim  in 


APPENDIX  471 

water  examination)  nutrient  gelatin  gives  a  relatively  larger  number  of  colonies  than 
distilled-water  gelatin,  nutrient  gelatin  should  be  used  when  one  gelatin  only  is 
employed.  At  the  same  time,  it  is  recognised  that  cultures  in  distilled-water 
gelatin  compared  with  cultures  in  nutrient  gelatin  often  give  useful  indications. 
Thus  with  an  unpolluted  water  the  number  of  colonies  is  usually  relatively  larger 
in  distilled-water  gelatin  than  in  nutrient  gelatin ;  with  a  polluted  water  the  con- 
verse is  the  case.  Therefore  the  use  of  both  gelatins  (distilled-water  and  nutrient) 
is  desirable,  sets  of  plates  being  made  with  each  medium. 

Similarly,  it  was  felt  by  many  members  of  the  committee  that  a  comparison  of 
the  ratio  of  the  number  of  organisms  developing  at  room  temperature  to  those 
developing  at  blood-heat  gives  useful  indications.  With  a  pure  water  this  ratio  is 
generally  considerably  higher  than  10  to  1,  with  a  polluted  water  this  ratio  is 
approached,  and  frequently  becomes  10  to  2,  10  to  3,  or  even  less.  The  actual 
number  of  organisms  growing  at  blood-heat  is  also  of  considerable  value  apart 
from  any  question  of  ratio.  Therefore  it  is  suggested  that  plates  of  nutrient  agar 
should  also  be  employed  and  incubated  at  blood-heat. 

In  certain  instances  it  is  true  that  this  ratio  may  be  unreliable.  Thus  with  surface 
waters,  especially  in  tropical  countries  (as  pointed  out  by  Major  Horrocks),  varieties 
of  the  B.  fluorescens  liquefaciens  and  non  liquefaciens  and  B.  liquefaciens  may  be 
abundant  and  grow  well  at  blood-heat. 

Preparation  and  Reaction  of  Media  for  Enumeration 

(a)  Distilled-Water  Gelatin. — Ten  per  cent,  gelatin  in  distilled  water,  and  brought 

to  a  reaction  of  +  10  (Eyre's  scale). 

(6)  Nutrient  Gelatin. — Ten  per  cent,  nutrient  gelatin,  preferably  made  with  meat 
(beef)  infusion  and  Witte's  peptone,  and  brought  to  a  reaction  of  +  10 
(Eyre's  scale). 

In  hot  weather  it  may  be  necessary  to  increase  the  percentage  of  gelatin. 
Some  members  of  the  committee  advocate  the  use  of  meat  extracts  in  place  of 
meat  infusion,  on  the  score  of  convenience  and  uniformity  of  composition,  Brand's 
Essence  being  recommended  as  the  best.     It  is  the  general  opinion,  however,  that 
Liebiy's  Extract  is  less  suitable  for  this  purpose. 

(c)  For  enumeration  at  blood-heat  it  is  recommended  that  nutrient  agar  should  be 

employed,  being  prepared  with  the  same  constituents  as  nutrient  gelatin, 
but  substituting  1J  per  cent,  of  powdered  agar  for  the  gelatin.  Reaction 
+  10. 

(d)  Distilled-Water  Agar. — Powdered  agar  1^  per  cent.,  dissolved  in  distilled 

water,  and  brought  to  a  reaction  of  +  10. 

Owing  to  the  changes  which  occur  in  the  reaction  of  the  medium  on  keeping,  the 
media  employed  should  preferably  be  not  more  than  three  weeks  old. 

Amounts  to  be  Plated,  Size  of  Dishes,  etc. — Gelatin. — For  an  ordinary  water 
amounts  of  0'2,  0'3,  and  0*5  c.c.  may  be  plated  in  Petri  dishes  of  not  less  than  10 
centimetres  diameter,  preferably  done  in  duplicate. 

Agar. — Two  plates  may  be  made  with  O'l  and  I'O  c.c.,  and  are  preferably 
duplicated. 

In  dealing  with  an  unknown  water,  and  in  all  cases  of  doubt,  additional  sets  of 
plates  should  be  prepared  with  a  dilution  of  the  water  (made  with  sterilised  tap- 
water)  often  or  hundred  fold,  according  to  circumstances. 

The  amount  of  the  medium  in  a  plate  should  be  10  c.c. 

The  sample  must  be  thoroughly  shaken  and  mixed  in  all  cases  before  plating. 

Temperature  of  Incubation. — (a)  Room  temperature  =  18-22°  C.  ;  (6)  blood-heat  = 
36-38°  C. 

Counting. — Counting  to  be  done  with  the  naked  eye,  preferably  in  daylight,  any 
doubtful  colony  being  determined  with  the  aid  of  a  lens  or  low-power  objective. 

Time  of  Counting.—  Gelatin  plates  should  be  counted  at  the  end  of  seventy-two 
hours  ;  but  in  all  cases  the  plates  should  be  inspected  daily,  in  order  that  the  count 
may  be  made  earlier  should  liquefaction  render  this  necessary. 

The  blood-heat  agar  plates  should  be  counted  at  the  end  of  forty  to  forty-eight 
hours. 


472  APPENDIX 


Search  for  B.  Coli 

Method. — The  committee  recommend  either — 

(a)  The  glucose-formate  broth  method  of  Pakes. 
(6)  The  bile-salt  broth  method  of  M'Conkey. 

Incubation  anaerobically  at  42°  C.  increases  the  chances  of  success  with  either 
medium,  and  is  strongly  recommended. 

It  has  also  been  suggested  that  the  neutral-red  (Griibler's)  glucose  broth  medium 
may  be  employed. 

The  committee  do  not  regard  with  favour  the  Parietti  method,  or  the  use  of 
carbolic  acid  media. 

Quantity  of  Water  to  be  Examined. — As  a  routine  50  c.c.  should  be  the  minimal 
quantity  examined  for  the  presence  of  the  B.  coli,  quantities  from  a  minimum  of 
O'l  c.c.  to  a  maximum  of  25  c.c.  being  added  to  the  tubes  of  culture  media. 

The  committee  are  of  opinion  that  it  is  preferable  to  add  the  water  directly  to  the 
tubes  of  culture  medium,  even  with  the  larger  amounts,  rather  than  first  to  concen- 
trate by  filtration  through  a  porcelain  filter  (the  filter-brushing  method).  The 
culture  media  recommended  may  be  diluted  with  at  least  an  equal  volume  of  the 
water  without  interfering  with  their  cultural  properties,  and  large  tubes  or  small 
flasks  may  be  used  for  the  larger  amounts. 

In  the  case  of  the  bile-salt-lactose-peptone  water,  the  medium  may  for  the  larger 
amounts  be  prepared  of  double  strength. 

Isolation  of  B.  coli,  if  Present. — If  indications  of  the  presence  of  the  B.  coli  be 
obtained  in  the  preliminary  cultivations,  the  organism  must  be  isolated  and  identified. 

This  may  be  done  by  making  surface  cultures  on  plates  of  either  (a)  litmus-lactose 
agar,  reaction  +10;  (6)  bile-salt  agar ;  (c)  nutrose  agar  of  Conradi  and  Drigalski ; 
or  (d )  ordinary  nutrient  gelatin. 

The  best  medium  of  all  is,  probably,  the  nutrose  agar  of  Conradi  and  Drigalski. 
Agar  media  have  the  advantage  of  saving  time. 

Identification  of,  and  Tests  for,  the  B.  coli. — Having  obtained  coli-like  colonies  on 
the  plates  made  from  the  preliminary  cultivations  of  the  water,  sub-cultures  must  be 
made  in  order  to  identify  the  organism.  The  following,  at  least,  should  be  made  : 

(a)  Surface  agar  at  37°  C.     The  abundant  growth  so  obtained  enables  many  sub- 
cultures and  preparations  to  be  made  if  required. 

eStab  and  surface  cultures  in  gelatine.     This  may  be  done  in  the  same  tube. 
Litmus  milk  incubated  at  37°  C. 
„  )  Glucose  litmus  medium. 

(«)  Lactose  litmus  medium. 

(/)  Peptone  water  for  indol  reaction. 

Characters  of  the  B.  coli. — The  B.  coli  is  a  small  motile,  non-sporing  bacillus, 
growing  at  37°  C.  as  well  as  at  room  temperature.  The  motility  is  well  observed  in 
a  young  culture  in  a  fluid  glucose  medium.  It  is  decolorised  by  Gram's  method  of 
staining.  It  never  liquefies  gelatin,  and  the  gelatin  cultures  should  be  kept  for  at 
least  ten  days  in  order  to  exclude  a  liquefying  bacillus.  It  forms  smooth,  thin 
surface  growths  and  colonies  on  gelatin,  not  corrugated,  growing  well  to  the  bottom 
of  the  stab  (facultative  anaerobe). 

It  produces  permanent  acidity  in  milk,  which  is  curdled  within  seven  days  at 
37°  C.  It  ferments  glucose  and  lactose,  with  the  production  both  of  acid  and  of  gas. 

The  typical  B.  coli  must  conform  to  the  above  description  and  tests. 

It  generally  also  forms  indol  (best  obtained  in  peptone-water  cultures),  gives  a 
thick  yellowish-brown  growth  on  potato  (greatly  dependent  on  the  character  of  the 
potato),  sometimes  (about  50  per  cent.)  ferments  saccharose,  changes  neutral-red 
(Grii bier's),  and  reduces  nitrates,  and  half  the  gas  produced  by  it  from  glucose  is 
absorbable  by  KOH  ;  and  these  tests,  if  time  and  opportunity  permit,  may  be  per- 
formed in  addition  to  the  foregoing. 

The  committee  recognise  that  atypical  B.  coli  are  me£  with,  but  in  the  present 
state  of  our  knowledge  hesitate  to  make  any  suggestion  with  regard  to  their 
significance. 


APPENDIX  473 

Streptococci 

The  committee  consider  that  it  is  a  distinct  advantage  to  search  for  streptococci. 
They  may  be  looked  for  by  making  hanging-drop  preparations  of  the  fluid  media 
employed  for  the  preliminary  cultivation  of  the  B.  coli  (glucose-formate  broth,  etc.). 
The  presence  or  absence  of  streptococci  in  these  tubes  gives  also  a  quantitative  value 
to  the  examination,  just  as  in  the  case  of  B.  coli,  and  the  result  obtained  should  be 
stated.  The  streptococci  should  be  isolated  (best  carried  out  on  nutrose  agar 
plates),  and  their  characters  determined. 

B.  Bnteritidis  Sporogenes 

As  already  stated,  the  committee  do  not  consider  that  it  is  essential  as  a  routine 
procedure  to  search  for  the  B.  enteritidis  sporoaenes,  though  in  certain  instances  it 
may  be  of  advantage  to  do  so.  A  negative  result  in  such  cases  is  probaby  of  more 
value  than  a  positive  one. 

This  report  is  the  outcome  of  prolonged  deliberations,  and  every  point  has  been 
carefully  considered  and  discussed  by  the  members  of  the  committee. 

In  conclusion,  the  committee  suggest  that  if  the  above  recommendations  were  to 
be  adopted  by  all  engaged  in  .the  bacteriological  examination  of  water  it  would 
conduce  to  uniformity  of  results,  and  would  render  comparable  the  data  obtained  by 
different  observers.  An  addendum  might  be  added  to  a  report  on  an  analysis 
conducted  on  these  lines,  to  the  effect  that  the  analysis  had  been  carried  out  in  con- 
formity with  the  procedures  recommended  by  the  committee  of  The  Royal  Institute 
of  Public  Health,  1904. 

The  committee  beg  to  acknowledge  their  great  indebtedness  to  Professor  R. 
Tanner  Hewlett,  M.D.,  D.P.H.,  upon  whom  the  great  burden  of  the  work  of  the 
committee  has  devolved. 

RUPERT   BOYCE,   M.B.,   F.R.S., 
Chairman. 

R.  TANNER  HEWLETT,  M.D.,  M.R.C.P., 
Hon.  Secretary. 

BACTERIOLOGICAL   EXAMINATION   OF   MILK* 

Physical  examination  (temperature,  reaction,  colour,  cream,  deposit, 
specific  gravity,  etc.)  of  the  milk  should  be  made  if  necessary.  The 
microscopical  examination  of  the  milk  before  and  after  centrifugalisation 
or  sedimentation  will  likewise  often  yield  useful  results. 

1.  Plate  Cultivation. — Dilute  as  required,  and  make  plate  cultivations  in 
Petri  dishes  or  flat-bottomed  flasks.     Six  or  more  gelatine  plates  should  be 
made  and  incubated  at  room  temperature.      Plates  should  also  be  made 
with  nutrient  agar  for  incubation  at  37°  C.     Other  media  may  also  be 
used.     The  plates  should  be  counted  on  the  second,  third,  and  fourth 
days,  and  the  necessary  sub-cultures  made.     Agar  plates  incubated  wholly 
at  18°  or  22°  C.,  will  in  the  long  run  show  more  colonies  than  when 
incubated  at  37°  C.  and  then  at  22°  C.  or  at  37°  C.  throughout. 

2.  Anaerobic  Cultivation. — At  the  same  time  that  the  primary  aerobic 
plate  cultivations  are  made,    similar  plates  should  be  made  on  lactose 
gelatine  and  lactose  agar  for  anaerobic  culture  (see  p.  117). 

3.  Primary  Tube  Cultivation. — Take  ten  tubes  of  10  c.c.  of  the  milk 

*  For  further  particulars  concerning  the  bacteriological  technique  in  milk 
examination,  see  Bacteriology  of  Milk,  by  Swithinbank  and  Newman,  1903,  pp. 
30-115. 


474  APPENDIX 

under  examination,  and  place  three  of  them  in  the  incubator  at  room 
temperature  and  three  of  them  at  37°  C.  Place  four  of  them  in  a  water 
bath  heated  to  80°  C.  for  fifteen  minutes,  and  then  enclose  each  of  the 
four  tubes  in  a  Buchner's  tube.  These  primary  cultures  may  be  tested 
in  forty-eight  hours  for  B.  coli,  the  presence  of  indol,  and  B.  cnteritidis 
sporogenes. 

4.  Secondary  or  Sub-cultures. — From   the   primary  cultivations,  make 
sub-cultures  on  selected  media  for  the  isolation  of  organisms  making 
their  appearance  on  the  plates,  or  what  is  often  preferable,  make  a  set 
of  plates  for  qualitative  examination  only. 

5.  Examination  for  Special  Micro-organisms. — The  milk  must  be  centri- 
fugalised  or  the  particulate  matter  allowed  to  gravitate  by  sedimentation. 
It  is,  as  a  rule,  useless  to  attempt  examination  microscopically  or  other- 
wise without  first  using  the   centrifuge   or   sedimentation   flask.     The 
deposit   is  then  to  be  stained  for  the   particular   organism    for    which 
search  is  being  made  (see  p.  476). 

For  centrifugalisation,  take  two  or  three  samples  of  the  milk  under 
examination  to  the  amount  of  about  40  c.c.  each,  and  place  it  in  the 
sterilised  tubes  of  the  centrifuge.  In  these  tubes  the  milk  may  be 
centrifugalised  for  ten  or  fifteen  minutes  at  3000  revolutions  a  minute. 
At  the  end  of  such  a  period  the  milk  in  each  tube  has  separated  into 
three  layers — at  the  top  there  is  a  dense  layer  of  cream,  at  the  bottom 
there  is  the  sediment  or  "slime"  containing  all  the  particulate  matter, 
between  these  two  is  the  separated  milk.  Aspirate  off  the  cream  by 
means  of  a  sterile  glass  tube  connected  with  an  aspirator  or  vacuum 
pump,  and  examine  separately ;  aspirate  all  the  separated  milk  except 
2  c.c.  The  remaining  sediment  is  so  compact  and  dense  that  the  tube 
may  now  be  inclined  and  the  sediment  fully  exposed  without  displace- 
ment. By  means  of  a  sterilised  platinum  loop  a  small  portion  may  be 
taken  up  and  spread  on  the  surface  of  half  a  dozen  slides,  and  stained. 
The  remainder  of  the  sediment  is  well  mixed  with  the  2  c.c.  of  milk  and 
used  for  inoculation  of  guinea-pigs. 

For  sedimentation,  take  two  conical  sedimentation  glasses  and  fill 
them  with  the  milk  under  examination,  allowing  them  to  stand  in  the 
refrigerator  for  twelve  to  fourteen  hours.  It  is  customary  to  add  a  few 
small  carbolic  crystals  to  each  flask.  On  the  completion  of  sedimenta- 
tion the  milk  has  separated  into  three  main  strata :  the  cream  at  the  top, 
the  sediment  at  the  apex  of  the  flask,  and  the  separated  milk  in  the 
middle.  The  cream  and  milk  may  then  be  carefully  decanted,  and  the 
sediment  will  be  available  for  examination. 

STAINING  METHODS  IN  MILK  EXAMINATION 

The  only  difficulty  which  presents  itself  in  the  preparation  of  milk  for 
the  microscope  is  the  simultaneous  staining  of  the  casein  and  fat  as  well 
as  the  organisms  which  may  seriously  confuse  the  issue.  Hence  the 
removal  of  the  two  former  substances  is  recommended,  as  follows  : — 

(a)  Staining  after  Clearing  with  5  per  cent.  Acetic  Acid. — The  slides  are 


APPENDIX  475 

thoroughly  cleaned  in  the  ordinary  way,  and  immediately  before  use  are 
again  washed  with  equal  parts  of  alcohol  and  ether.  Several  loopfuls  of 
the  milk  to  be  examined  are  now  placed  on  the  slide  and  allowed  to  dry 
at  the  temperature  of  the  room,  being  protected  from  the  air  by  means 
of  a  small  glass  cover.  When  the  film  is  dry  it  is  fixed,  preferably  with 
alcohol  and  ether,  as  described  below.  It  is  then  washed  alternately 
with  a  5  per  cent,  solution  of  acetic  acid  and  distilled  water  until  there 
is  but  little  apparent  film  left  upon  the  slide,  which  is  then  dried 
between  layers  of  fine  filter-paper.  The  specimen  may  now  be  stained 
by  means  of  any  of  the  ordinary  aniline  dyes,  washed  in  distilled  water, 
again  dried,  and  examined  under  the  microscope.  Ether,  chloroform, 
various  strengths  of  alcohol,  and  other  clearing  agents  may  be  used  if 
preferred. 

(6)  Saponification. — If  it  be  desired  to  retain  the  background  of  casein 
and  fat,  it  will  be  found  best  to  saponify  the  milk  in  the  following 
manner : — Prepare  the  film  of  milk  as  before,  but  before  drying  it  add 
an  equal  number  of  loopfuls  of  a  sodium  carbonate  or  sodium  hydrate 
solution  (5  per  cent,  to  50  per  cent,  dilution).  The  loopfuls  of  milk  and 
soda  solution  should  be  placed  in  immediate  proximity  to  each  other  on 
the  slide,  and  thoroughly  mixed  by  means  of  the  platinum  loop.  By 
this  means  an  even  distribution  of  the  bacteria  is  obtained.  The  film  is 
then  dried  by  gentle  heating,  stained,  washed,  and  cleared  with  xylol. 
The  result  will  be  that  the  organisms  will  be  stained  more  deeply  in 
colour  than  the  background  of  saponified  matter. 

(c)  Clearing  with  Acetic  Acid  after  Saponification. — The  best  prepara- 
tions are  obtained  by  a  combination  of  the  above  methods.  For  this 
purpose  the  films  are  prepared  exactly  as  in  the  ordinary  Saponification 
method  above  described,  but  as  soon  as  the  films  have  become  saponified, 
instead  of  at  once  proceeding  to  stain  with  the  desired  dye,  the  film  is 
thoroughly  cleared  by  several  alternate  washings  with  the  5  per  cent, 
solution  of  acetic  acid  and  distilled  water.  The  subsequent  procedure 
is  as  in  («). 

Methods  Of  Fixation. — The  object  of  fixing  is  to  coagulate  the 
albuminous  material,  and  cause  perfect  adhesion  of  the  prepared  film  to 
the  slide.  The  following  alternative  methods  are  recommended : — 

(a)  Heat. — Holding  the    glass  slide  by  one  extremity  between  the 
thumb  and  index  finger  of  the  right  hand,  pass  it,  film  side  upwards, 
gently  through  the  flame  three  times,  allowing  the  under  surface  to  rest 
on  the  back  of  the  left  hand  between  each  passage. 

(b)  Alcohol-ether. — Place  one  or  two  drops  of  a  mixture  of  equal  parts 
of  absolute  alcohol   and    ether   upon  the    dried    film,  and  allow  it   to 
evaporate. 

(c)  Formal-alcohol. — Formalin  1  part,  absolute  alcohol  9  parts.     Leave 
in  contact  for  from  three  to  four  minutes,  wash  well  in  water,  blot  off 
excess  of  moisture,  and  stain. 

(cT)  Perchloride  of  Mercury. — Saturated  aqueous  solution.  Leave  in 
contact  with  the  film  for  four  or  five  minutes.  Wash  off  with  a  stream 
of  water,  and  apply  Gram's  iodine  solution  in  order  to  dissolve  out  any 


476  APPENDIX 

formed  crystals  of  the  salt.  Wash  again  in  water,  blot  off  excess  of 
moisture,  and  apply  stain.  This  fixing  agent  should  be  used  on  all 
occasions  when  dealing  with  morbid  material  or  cultures  of  a  specially 
virulent  nature. 

METHODS  OF  EXAMINATION  FOR  SPECIAL  MICRO-ORGANISMS  IN  MILK 

Bacillus  pseudo-tuberculosis  Of  Pfeiffer  (found  in  London  milk 
by  K/eiri). — By  the  centrifuge  or  by  sedimentation  in  an  ice-chest  for 
twenty-tour  hours,  obtain  the  particulate  matter  of  the  milk  to  be 
examined.  Inoculate  2  c.c.  into  a  guinea-pig  subcutaneously  or  intra- 
peritoneally.  In  the  course  of  three  to  four  weeks  caseo-puruleiit 
nodules  will  occur  in  the  inguinal  glands  (if  subcutaneously  inoculated), 
or  in  the  omentum  and  pancreas  and  other  organs  (if  intra-peritoiieally). 
Cultures  may  be  obtained  best  from  glands,  spleen,  pancreas,  or  liver. 
Examine  the  nodules  by  staining  and  culture.  They  will  have  the 
following  characters  if  the  disease  be  pseudo-tuberculosis  :  (a)  Absence 
of  giant  cells  ;  (6)  absence  of  the  true  tubercle  bacillus  ;  (c)  presence  of 
large  numbers  of  B.  pseudo-tuberculosis  ;  and  (d)  signs  of  a  rapid  and  not 
a  slow  development. 

Method  of  Staining. — Make  films  in  the  ordinary  way  and  stain  with 
Loffler's  methylene-blue,  heating  the  stain  till  it  steams  (Klein).  Wash 
in  distilled  water.  Nodules  may  be  hardened  in  Miiller's  fluid  and 
spirit,  and  sections  cut  and  stained  by  placing  in  Loffler's  blue  for 
twenty -four  hours  and  counter-staining  in  a  mixture  of  eosin  and 
methylene-blue.  Loffler's  blue  may  also  be  used  for  staining  the 
bacillus  in  milk-films  made  from  sediment.  Gram's  method  is  also 
applicable,  but  the  bacillus  is  not  acid-fast,  and  will  not  hold  the  Ziehl- 
Neelsen  stain. 

Bacillus  diphtherias. — By  centrifuge  or  sedimentation  obtain  the 
particulate  matter  of  the  milk  under  examination  and  inoculate  it  into  a 
guinea-pig.  Sub-culturing  from  the  tissues  of  the  guinea-pig,  or,  having 
obtained  sediment  as  above,  inoculate  six  tubes  or  plates  of  Loffler's 
medium  (ox  serum  3  parts,  veal  broth  1  part — the  broth  to  contain 
glucose  1  per  cent.,  peptone  1  per  cent.,  and  sodium  chloride  0'5  per 
cent.).  Upon  this  medium  the  Klebs-Loffler  bacillus  grows  rapidly  in 
twelve  to  twenty  hours,  producing  scattered  nucleated,  round,  white 
colonies  which  later  become  yellow. 

Method  of  Staining. — Gram's  method  as  modified  by  Nicolle  (see 
p.  459)  will  be  found  the  most  satisfactory,  but  the  methylene-blue 
solution  of  Loffler  is  often  used.  This  consists  of  30  c.c.  of  a  saturated 
alcoholic  solution  of  methylene-blue  added  to  100  c.c.  of  a  '01  per  cent, 
solution  of  caustic  potash.  By  this  stain  the  striped  appearance  of  the 
bacilli  of  older  cultures  on  blood  serum  is  obtained  more  readily  than  by 
other  methods. 

Neissers  Method  for  Differentiation  of  the  Diphtheria  Bacillus. — This 
method  consists  in  applying  two  stains  as  follows.  Stain  I.  is  made  of 
1  gramme  of  methylene-blue  dissolved  in  20  c.c.  of  a  95  per  cent,  alcohol, 


APPENDIX  477 

and  950  c.c.  of  distilled  water.  To  this  is  added  50  c.c.  of  glacial  acetic 
acid.  Stain  II.  consists  of  2  grammes  of  vesuvin  dissolved  in  1000  c.c. 
of  boiling  distilled  water.  Both  stains  are  filtered  before  use.  Prepare 
films  in  usual  way,  and  stain  with  No.  I.  for  thirty  seconds.  Wash  in 
water  and  then  stain  with  No.  II.  for  thirty  seconds.  Wash,  dry, 
and  mount.  The  bacilli  are  stained  brown  by  vesuvin  and  the  meta- 
chromatin  granules  blue-black.  Some  bacteriologists  place  great 
reliance  upon  the  diagnostic  value  of  Neisser's  stain  for  the  diphtheria 
bacillus  from  blood  serum  cultures  and  from  swabs.  In  the  latter  case 
the  stain  is  sometimes  used  as  a  "  rapid  method  of  diagnosis."  It  is  not, 
however,  absolutely  reliable. 

StreptOCOCCUS  in  Milk. — By  centrifuge  or  sedimentation  obtain 
the  particulate  matter  of  the  milk.  Take  a  sterilised  platinum  loop,  dip 
in  the  sediment,  and  remove  a  drop  of  it.  Distribute  this  in  a  test- 
tube  containing  1  to  2  c.c.  of  sterile  salt  solution.  Inoculate  agar  plates 
with  a  drop  of  this  dilution,  and  incubate  at  37°  C.  When  the  colonies 
appear,  sub-culture  those  resembling  streptococcus  colonies  in  bouillon, 
and  on  blood  serum.  Sub-culture  from  the  bouillon  in  milk,  gelatine, 
and  agar,  carefully  noting  the  characters  of  the  growth,  etc.  Or 
guinea-pigs  may  be  inoculated  in  the  subcutaneous  tissue  of  the 
groin  or  intra-peritoneally.  An  acute  purulent  inflammation  will  be 
set  up  in  the  exudation  of  which  streptococcus  will  occur  in  large 
numbers. 

Method  of  Staining, — Gram's  method  is  the  most  satisfactory.  Next  to 
Gram's  stain  the  most  useful  is  Loffler's  blue.  It  may  be  noted  that 
most  of  the  putrefactive  organisms  do  not  hold  Gram's  stain. 

Bacillus  COli  COmmuniS. — («)  Dilute  the  milk  to  be  examined  500 
or  1000  times.  Take  a  sterilised  brush,  dip  it  in  the  dilution,  and  brush 
over  the  surface  of  six  agar  plates  without  recharging  the  brush. 
Incubate  at  42°  C.,  and  sub-culture  the  coliform  colonies  (bouillon,  milk, 
litmus  milk,  gelatine  "  shake  "  cultures,  bile-salt-glucose-peptone,  etc.). 

(/;)  Take  six  tubes  of  phenol  bouillon  (0*05  per  cent,  of  carbolic  acid), 
and  inoculate  them  with  crude  or  diluted  milk.  Those  which  show 
abundant  turbidity  after  twenty-four  to  forty-eight  hours  at  37°  C.  may 
be  plated  out  on  phenol  gelatine,  incubated  at  20°  C.,  and  the  coli 
colonies  sub-cultured ;  or  diluted  milk  may  be  at  once  plated  out  on 
phenol  gelatine,  and  colonies  sub-cultured  on  such  media  as  will  show  the 
characteristics  of  the  organisms. 

The  main  characters  of  the  B.  coli  group  of  organisms  may  be  briefly 
restated  here,  though  particulars  will  be  found  elsewhere  in  the  present 
volume  :— (1)  They  are  non-sporing  and  non-liquefying  ;  (2)  they  rarely 
stain  by  Gram's  method  ;  (3)  they  are  motile ;  (4)  they  produce  acid  and 
gas  in  glucose  and  lactose  media ;  (5)  they  produce  acid  in  milk,  and 
usually  coagulate  it ;  (6)  they  grow  well  at  a  temperature  of  42°  C. 
Referring  to  the  isolation  of  B.  coli,  Houston  writes :  "  No  test  based  on 
observation  of  a  change  or  changes  produced  in  the  nutrient  medium, 
and  supposed  to  be  characteristic  of  B.  coli,  can  compare  with  isolation 
from  plate  cultivations  of  the  microbes  suspected  to  be  B.  coli,  and  the 


478 


APPENDIX 


subsequent  attentive  study  of  the  biological  characters  of  pure  cultures 
of  these  bacteria  grown  in  various  media.  "* 

Bacillus  enteritidis  sporogenes  of  Klein. — Take  six  tubes  con- 
taining 15  c.c.  of  fresh  milk  and  sterilise  them  by  boiling  for  half  an 
hour.  Rapidly  cool  them  by  placing  them  in  a  beaker 
of  cold  water,  add  to  each  tube  1  c.c.  of  a  1  in  500 
dilution  of  the  milk  to  be  examined,  or  if  it  be  pre- 
ferred 0*1  c.c.  of  the  crude  milk.  Heat  the  inocu- 
lated tubes  at  80°  C.  for  fifteen  minutes.  Then  remove 
and  cool,  and  place  in  Buchner  tubes  or  cylinder  con- 
taining freshly  prepared  mixture  of  pyrogallic  acid  and 
potassium  hydrate  solution.  Seal  the  Buchner  tubes 
or  cylinder  with  great  care,  making  it  absolute.  Place 
the  Buchner  apparatus,  including  the  milk  tubes,  in  the 
incubator  at  37°  C.  After  forty-eight  hours  take  out 
the  tubes,  and  examine  them  for  the  B.  enteritidis 
sporogenes.  If  necessary,  inoculate  guinea-pigs  sub- 
cutaneously  with  1  c.c.  of  the  whey,  which  in  a  few 
hours  causes  swelling  at  the  point  of  inoculation,  and 
extensive  gangrene  of  the  subcutaneous  and  muscular 
tissues  with  sanguineous  exudation  ;  the  animal  dies  in 
twenty-four  or  thirty  hours.  The  B.  butyricus  of  Botkin 
may  produce  similar  changes  in  milk  tubes,  but  it  has 
no  pathogenic  action.  Milk  may  be  examined  directly 
by  placing  20  c.c.  in  tubes  and  treating  as  above.  For 
the  "euteritidis  change"  in  the  milk,  see  p.  307. 

Bacillus  tuberculosis. — Obtain  the  sediment  of 
the  milk  under  examination  and  inoculate  2  c.c.  of  it 
into  the  subcutaneous  tissue  of  the  guinea-pig.  In 
about  four  weeks'  time,  local  if  not  general  tubercu- 
losis will  have  been  set  up.  Take  some  of  the  dis- 
charge and  stain  it  after  the  Ziehl-Neelsen  method. 
The  sediment  of  tuberculous  milk  may  be  stained 
forthwith,  without  inoculation,  by  the  same  method, 
and  in  some  cases  the  tubercle  bacillus  may  be  thus 
detected,  but,  generally  speaking,  the  only  sure  test 
is  inoculation  of  animals.  The  pathological  process  is 
slower  than  in  pseudo-tuberculosis,  and  on  exami- 
nation the  diseased  tissues  show  giant  cells  and 
form  of  Buchner  Tub?  numerous  tubercle  bacilli  arranged  within  the  giant  cell 

(see  Plate  23,  p.  328). 

Method  of  Rabinowitsch  for  Tubercle  Bacillus  in  Butter. — The  butter 
is  placed  in  sterile  conical  glass  in  the  incubator  at  37°  C.,  where  on 
melting  it  will  arrange  itself  in  two  layers.  Three  c.c.  of  the  superna- 
tant fatty  liquid  are  injected  into  the  peritoneal  cavity  of  a  guinea- 
pig.  A  similar  quantity  of  the  deposit  is  treated  in  a  like  manner, 


Second  Report  of  Royal  Commission  on  Sewage  Disposal,  1902,  p.  411. 


APPENDIX  479 

and  finally,  two  or  three  other  animals  are  inoculated  intra-peritoneally 
with  the  semi-liquid  substance  obtained  on  mixing  together  the  two 
layers  into  which  the  butter  was  formed.  At  the  end  of  expiration  of 
seventy  days  the  animals  which  have  not  already  succumbed  are 
sacrificed,  and  a  careful  post-mortem  examination  is  made.  Microscopic 
preparations  and  cultures  are  made  from  any  organs  affected.  The 
latter,  taken  together  with  the  general  aspect  of  the  lesions,  will,  in  the 
majority  of  cases,  be  sufficient  to  enable  a  diagnosis  to  be  made  between 
the  true  bacilli  of  tuberculosis,  and  other  acid-fast  organisms  resembling 
it.  Bacilli  which  resists  in  a  moderate  or  somewhat  feeble  manner 
decolorisation  by  acids,  which  develop  rapidly  at  a  temperature  of  37°  C., 
and  grow  feebly  at  ordinary  room  temperature,  which  exhibit  chromo- 
genic  properties  in  culture,  and  give  rise  in  the  guinea-pig  to  lesions 
which  are  not  characteristically  those  of  tuberculosis,  must  be  regarded 
as  organisms  of  the  acid-fast  group,  non-pathogenic  for  man,  though 
possibly  related  in  some  degree  to  the  true  bacillus  of  tuberculosis  (see 
also  p.  358). 

Another  method  is  that  indicated  by  Roth.  Five  grammes  of  butter 
are  vigorously  shaken  up  in  sterile  water,  and  the  whole  is  then  centri- 
fugalised.  A  fat-free  deposit  is  thus  obtained,  and  given  quantities  of 
this  are  injected  into  animals  in  the  ordinary  manner. 

SPECIAL  METHODS 

Examination  Of  Colostrum. — Colostrum  is  the  term  applied  to  the 
first  milk  yielded  by  the  cow  after  parturition.  It  differs  considerably 
from  ordinary  milk,  and  generally  appears  as  a  thick,  turbid,  yellowish, 
viscid  fluid.  When  examined  under  the  microscope,  it  is  found  to  con- 
tain, in  addition  to  the  ordinary  milk  corpuscles,  peculiar  conglomera- 
tions of  very  minute  fat  granules  which  are  hence  known  as  colostrum 
corpuscles.  The  chief  chemical  differences  between  colostrum  (or 
beastings)  and  milk  are  mainly  three.  First,  colostrum  is  deficient  in 
casein.  Secondly,  it  is  proportionately  rich  in  albumen.  Thirdly,  it  con- 
tains nearly  three  times  more  salts  than  milk.  Probably  it  is  this  excess 
of  salts  that  usually  causes  it  to  exert  a  purgative  effect  upon  the  new- 
born calf,  and  thus  to  remove  the  meconium  which  has  accumulated  in 
the  foetal  intestine. 

The  difficulties  of  bacteriological  examination  of  such  a  subject  as 
colostrum  are  considerable.  At  the  outset,  a  fair  sample  is  only  obtain- 
able by  adopting  the  following  precautions :  (a)  The  teats  and  udder  to 
be  cleansed  ;  (b)  milking  to  be  carried  out  as  soon  after  calving  as  pos- 
sible, when  the  calf  has  sucked;  (c)  the  first  part  of  the  "milking"  to 
be  discarded,  and  the  last  part  only  to  be  examined.  When  the 
colostrum  reaches  the  laboratory,  it  must  be  diluted  in  precisely  the 
same  manner  as  thick  cream.  After  abundant  dilution  treat  the  solution 
in  the  ordinary  way,  by  staining  preparations  for  the  microscope,  plating 
out  on  various  media,  and  sub-culturing. 

Bacteriological  Examination  of  Butter.— Take  a  quarter  of  a 


480  APPENDIX 

pound  of  butter  and  place  it  in  a  sterilised  flask  with  150  c.c.  of  sterile 
salt  solution.  Place  the  flask  in  the  water  bath  at  about  35°  C.,  and 
shake  gently  until  the  butter  has  melted.  The  contents  of  the  flask  now 
appear  as  a  milk-like  emulsion.  A  small  quantity  of  this  mixture  may 
be  used  for  plate  cultivation  on  gelatine  and  agar,  as  in  milk.  The 
remainder  should  be  placed  in  a  sedimentation  flask  in  the  refrigerator 
for  twenty-four  hours.  By  this  means  the  particulate  matter  of  the 
butter,  including  the  contained  organisms,  are  deposited.  After  remov- 
ing the  superficial  solidified  fat  by  means  of  a  sterile  spatula,  the  turbid 
fluid  may  be  decanted,  and  the  sediment  collected  for  microscopical 
examination  or  the  injection  of  guinea-pigs. 

Examination  Of  Cheese.— With  a  knife  previously  sterilised  by 
pissing  through  the  flame,  cut  off  from  the  piece  of  cheese  under 
examination  a  thin  slice  parallel  to  the  surface.  Remove  this,  and 
with  a  second  sterile  knife  cut  perpendicularly  downward  from  the 
bared  surface.  Pass  down  into  the  latter  cut  a  coarse  sterile  platinum 
needle,  of  which  a  small  portion  near  the  extremity  has  been  slightly 
roughened  with  a  file. 

Inoculate  with  this  needle  a  sufficient  number  of  tubes  of  bouillon 
from  which  plate  cultivations  can  subsequently  be  made  for  isolation 
purposes,  and  placed  under  both  aerobic  and  anaerobic  conditions. 

Examination  of  Milk  for  Pus  Cells.— Place  10  c.c.  of  the  milk  to  be 

examined  in  each  tube  of  the  centrifuge  (Plate  5,  p.  74)  and  centrifugalise 
for  two  minutes.  Pour  off  the  supernatant  fluid,  and  with  a  sterilised 
needle  or  pipette  take  up  a  small  quantity  of  the  sediment  remaining  in 
the  tube.  Spread  the  sediment  evenly  over  the  surface  of  an  ordinary 
glass  slide,  and  dry  over  the  flame  of  a  Bunsen  burner  or  on  the  drying 
stage.  Wash  the  fixed  film  with  ether  (or  alternately  with  absolute 
alcohol  and  ether)  until  all  the  superfluous  fat  is  removed,  and  stain. 
The  preparation  may  be  stained  (a)  by  one  of  the  ordinary  solutions 
such  as  Loffler's  blue,  etc. ;  or  (6)  by  Gram's  method.  Examine  under 
the  microscope  with  a  y^th  oil  immersion  lens. 

Inoculation  of  Guinea-pig's  in  Milk  Examination. — It   will 

be  sufficient  to  remark  that  the  simplest  forms  of  inoculation  are  all  that 
are  usually  required  in  milk  investigation,  namely,  the  infra-peritoneal  and 
subcutaneous.  In  some  cases  it  may  be  sufficient  to  inoculate  a  few  c.c.  of 
the  original  milk ;  but,  .as  a  rule,  it  is  advisable  to  centrifugalise,  or  use 
the  sedimentation  flask  containing  about  250  c.c.  From  the  deposit  or 
sediment  two  guinea-pigs  may  be  inoculated,  the  one  subcutaiieously  in 
the  groin,  the  other  intra-peritoneally.  Particularly  is  this  necessary  in 
making  a  reliable  and  exhaustive  search  for  the  B.  tuberculosis.  Micro- 
scopic examination  alone  for  this  organism  is  not  reliable  (see  p.  478). 
The  details  of  the  process  as  carried  out  in  practice  are  as  follows  : — 

After  centrifugalisation  the  deposit  is  mixed  with  the  2  c.c.  of  milk 
remaining  in  the  tube  after  aspiration  of  that  which  is  superfluous.  Two 
guinea-pigs  (of  say  250  grammes  weight  each)  are  taken  and  inoculated 
with  the  deposit  from  about  40  c.c.  of  milk.  The  fluid  is  inoculated 
subcutaneously  on  the  inner  side  of  the  leg  under  strict  aseptic  precau- 


APPENDIX  481 

tions  (the  skin  having  been  washed  with  1-1000  corrosive  sublimate,  and 
shaved).  In  less  than  a  fortnight's  time,  if  the  inoculated  milk  contained 
a  considerable  number  of  tubercle  bacilli,  typical  infection  of  the 
popliteal  and  inguinal  glands  can  be  detected.  If  the  milk  contained 
very  few  bacilli  the  infection  is  much  slower  (fifth  week).  After  the 
animal  has  been  killed  the  presence  of  the  tubercle  bacilli  can  be 
detected  in  the  inguinal  glands  and  the  spleen.  Some  workers  make  it  a 
rule  to  inoculate  two  guinea-pigs  from  the  sediment  of  the  milk,  one 
receiving  half  of  the  sediment  subcutaneously  in  the  groin,  the  other 
receiving  the  other  half  intra-peritoneally. 

BACTERIOLOGICAL   DIAGNOSIS   IN   SPECIAL   DISEASES 

1.  Diphtheria. — Obtain  a  piece  of  the  membrane  or  a  "  swab  "  from 
the  throat.  Take  a  piece  of  stout  iron  wire  and  twist  a  piece  of  cotton 
wool  round  one  end  of  it,  and  insert  in  a  test-tube,  and  sterilise.  By 
means  of  such  a  swab  obtain  a  rubbing  of  the  suspected  throat.  Then 
scraping  off  from  the  swab  sufficient  material  for  (a)  a  microscopic 
examination,  (6)  smear  the  swab  over  the  surface  of  agar  and  blood 
serum  media,  and  finally  (c)  place  in  a  tube  of  sterilised  broth.  Thus 
we  have  material  for  a  film  preparation,  for  cultivation,  and  for  animal 
inoculation.  Make  the  film  in  the  usual  way,  and  stain  with  Nicolle's 
modification  of  Gram  (see'  p.  458)  or  Neisser's  stain  (see  p.  476). 
Examine  under  the  microscope.  The  value  of  examining  such  a  prepara- 
tion microscopically  depends  upon  the  experience  of  the  bacteriologist. 

Of  culture  media,  blood  serum  is  perhaps  the  best,  but,  if  no  serum 
tubes  can  be  had,  an  egg  may  be  used.  It  should  be  boiled  hard,  the 
shell  chipped  away  from  one  end  with  a  knife  sterilised  by  heating,  and 
the  inoculation  made  on  the  exposed  white  surface ;  the  egg  is  then 
placed,  inoculated  end  downwards,  in  a  wine-glass  of  such  a  size  that  it 
rests  on  the  rim  and  does  not  touch  the  bottom.  A  few  drops  of  water 
may  with  advantage  be  put  at  the  bottom  of  the  glass  to  keep  the  egg 
moist.  The  preparation  is  kept  in  a  warm  place  for  twenty-four  to 
forty-eight  hours,  and  then  examined.  The  examination,  of  course, 
consists  in  staining  and  preparing  specimens  for  the  microscope,  and 
observing  the  form,  arrangement,  and  characters  of  the  organism  or 
organisms  present.  The  same  is  done  for  cultures  on  agar  or  blood  serum. 
On  the  latter  the  colonies  show  characteristic  growth.  A  small  piece  of 
the  membrane  may  be  detached,  washed  in  water,  and  stained  for 
the  bacilli. 

To  differentiate  the  true  or  Klebs-Loffler  bacillus  from  the  pseudo 
or  Hofmann  bacillus,  note  especially  that  Hofmann's  bacillus  is  plumper, 
shorter,  and  thicker  in  the  middle  than  the  true  diphtheria  bacillus.  It 
also  stains  more  regularly,  grows  better  on  alkaline  potato,  and  produces 
an  alkaline  reaction  in  neutral  litmus  agar  or  bouillon  incubated  for  two 
days  at  37°  C.  It  is  non-pathogenic  for  guinea-pigs,  whereas  the  Klebs- 
Loffler  bacillus  is  pathogenic. 

2.  Tetanus. — The  detection  of  the   bacillus  of  tetanus  in  the  dis- 

2  H 


482  APPENDIX 

charge  of  a  tetanic  wound  is  not  always  easy.  Make  preparations,  and 
stain  with  carbol-fuchsin.  Drumstick-shaped,  spore-bearing  bacilli  are 
to  be  looked  for.  If  a  small  piece  of  tissue  is  available,  sections  should 
be  prepared  and  double-stained.  Cultivations  should  also  be  made  from 
the  discharge  in  blood  serum  or  glucose  agar  incubated  at  37°  C.  for 
forty-eight  hours.  Then  keep  the  culture  at  80°  C.  for  twenty  to  thirty 
minutes,  to  kill  all  non-sporing  bacilli.  Sub-culture  in  glucose  gelatine 
in  hydrogen  at  22°  C.,  and  examine  in  five  days.  Animal  inoculation 
(mice  and  guinea-pigs)  is  generally  necessary. 

To  isolate  the  tetanus  bacillus  from  soil,  proceed  as  follows : — Make 
an  emulsion  of  the  soil  in  sterilised  water.  Expose  it  to  80°  C.  for 
twenty  minutes.  Add  1  c.c.  of  the  emulsion  to  each  of  three  tubes  of 
glucose-formate  broth,  and  incubate  anaerobically  in  Buchner's  tubes  at 
37°  C.  After  twenty-four  hours'  incubation,  inoculate  guinea-pigs  sub- 
cutaneously,  using  Ol  c.c.,  and  observe  results.  Also  make  glucose-agar 
plates  from  the  same  emulsion  (after  heating  to  80°  C.),  and  incubate 
anaerobically  in  Bulloch's  apparatus. 

3.  Tuberculosis. — The  tubercle  bacillus  is  an  acid-fast  organism, 
stained  by  Ziehl-Neelsen  method.     But  several  allied  organisms  possess 
the  same  tinctorial  properties,  and  therefore  inoculation  into  a  guinea-pig 
is  frequently  necessary  for  diagnosis.     Sputum,  however,  is  generally 
accepted  as  proved  to  be  tubercular  if  bacilli  having  the  morphology  and 
staining  properties  of  the  tubercle  bacillus  are  present. 

4.  Typhoid. —  Widal's  Application  ofGrubers  Reaction.  This  diagnostic 
test  depends  upon  the  effect  which  the  blood  serum  of  a  person  suffering 
from  typhoid  fever  has  upon  the  B.   typhosus.     The  effect  is  twofold. 
In  the  first  place,  the  actively  motile  B.  typhosus  becomes  immotile ;  and 
secondly,  there  is  an  agglutination,  or  grouping  together  in  colonies,  of 
the  B.  typhosus.     Neither  of  these  features  occur  if  healthy  human  blood 
serum  is  brought  into  contact  with  a  culture  of  the  typhoid  bacillus. 

The  method  of  using  the  test  is  as  follows  : — 

(a)  Collection  of  Serum. — Wash  the  lobe  of  the  patient's  ear  with 
antiseptic  (2  per  cent,  lysol),  and  by  rubbing  render  the  ear  hyperaBmic. 
Wash  with  methylated  spirit  and  dry.  Puncture  the  vein  of  the  lobe 
with  a  sterilised  needle  or  lancet,  and  collect  the  issuing  blood  in  a 
pipette.  Hold  one  end  in  contact  with  the  bleeding  point,  and  lower  the 
other  end.  By  gravity  the  blood  will  enter  the  pipette ;  if  not,  gentle 
suction  may  be  applied.  When  full  to  the  shoulder,  remove  the  pipette, 
and  placing  the  clean  end  to  the  lips,  draw  the  blood  gently  but  com- 
pletely into  the  body  of  the  pipette.  Now  seal  the  ends  in  a  flame,  and 
let  the  pipette  lie  horizontally  till  the  blood  is  coagulated. 

(6)  Dilution  of  Serum. — Place  the  pipette  in  the  vertical  position, 
preferably  in  an  ice-chamber,  and  in  a  few  hours  the  clear  serum,  free 
from  corpuscles,  will  collect  at  the  lower  end,  ready  for  dilution.  If 
necessary,  centrifugalise  to  obtain  corpuscle-free  serum.  There  are 
several  methods  of  dilution  used  in  practice,  but  broadly  they  are 
divisible  into  two,  a  rough-and-ready  dilution  and  an  exact  measured 
dilution. 


APPENDIX  483 

The  rough  dilution  is  to  take  of  the  corpuscle-free  serum  to  be 
examined  one  drop.  Dilute  it  with  nine  parts  of  neutral  bouillon. 
Mix  on  a  slide  or  cover-glass  a  drop  of  this  one-tenth  diiutiou  of 
serum  one  or  more  drops  of  typhoid  broth  cultivation  of  eighteen  to 
twenty-four  hours'  growth.  The  serum  and  culture  are  thoroughly 
mixed  together  in  the  trough  of  a  hollow-ground  slide,  and  a  single  drop 
is  taken,  placed  upon  an  ordinary  clean  slide,  and  a  cover-glass  super- 
imposed ;  or  the  mixture  may  be  made  on  the  cover-glass  and  super- 
imposed on  the  slide. 

The  measurement  method  is  to  dilute  the  serum  by  exact  quantities, 
giving  say,  a  10  per  cent.,  a  1  per  cent.,  and  a  O'l  per  cent,  dilution  ;  or 
three  mixtures  containing  respectively  50  per  cent.,  5  per  cent.,  and 
0*5  per  cent,  of  serum.  The  50  per  cent,  dilution  is  made  by  adding 
equal  loopfuls  of  serum  and  of  a  typhoid  broth  culture  on  a  slide  or 
cover-slip.  The  5  per  cent,  is  made  by  diluting  10  c.m.  (measured  by 
graduated  haematocytometer)  of  the  serum,  with  90  c.m.  of  the  broth 
culture  in  a  small  sterilised  test-tube.  After  thoroughly  mixing,  one 
loopful  of  this  dilution  (now  10  per  cent.)  is  mixed  with  one  of  cultivation. 
The  0-5  per  cent,  is  made  by  first  diluting  10  c.m.  of  the  10  per  cent, 
serum  with  90  c.m.  of  sterile  broth  in  a  small  test-tube,  and  then  mixing 
equal  loopfuls  of  this  diluted  serum  and  of  the  broth  culture. 

(c)  The  Typhoid  Culture  used  should  be  one  sub-cultured  from  a  virulent 
culture,  and  should  be  a  broth  or  agar  culture  of  about  eighteen  to  twenty- 
four  hours ;  and,  if  preferred,  may  be  filtered  before  use  to  remove  any 
normally  agglutinated  masses  of  bacilli  before  commencing  the  test. 

(d)  The  Reaction. — The  reaction  is  positive  if  the  bacilli  have  become 
grouped  together  tightly  into  clumps  (agglutination),  leaving  the  field 
between  the  clumps  free  from  bacilli.     Immotility  will  also  be  present. 
The  reaction  time  is  half-aii-hour  (see  Plate  20).     In  his  first  experiments, 
Widal  used  a  test-tube  in  the  following  manner  : — The  blood  to  be  tested 
is  diluted  by  one  part  of  it  being  added  to  fifteen  parts  of  broth  in  a 
test-tube.     The  mixture  is  inoculated  with  a  drop  of  a  typical  B.  typhosus 
culture.     The  tube  is  then  incubated  at  37°  C.  for  twenty-four  hours, 
after  which  it  is  examined.     If  the  reaction  be  positive,  the  broth  appears 
comparatively  clear,  but  at  the  bottom  of  the  test-tube  a  more  or  less 
abundant  sediment  will  be  found.     This  is  due  to  the  clumps  of  bacilli 
having  fallen  owing  to  gravity.     If,  on  the  other  hand,  the  reaction  is 
negative,  the  broth  will  appear  more  or  less  uniformly  turbid.     This 
method  is  not  as  satisfactory  as  the  one  described. 

Some  bacteriologists  use  two  dilutions,  1  in  20  and  1  in  40,  with  a 
time  limit  of  one  hour  for  each  case.  The  reactions  obtained  are 
interpreted  as  follows  : — Where  both  dilutions  show  clumping  and  loss  of 
motility  at  the  end  of  the  hour  a  diagnosis  of  "enteric  fever"  is  made  ; 
but  if  the  reaction  is  present  only  in  the  1  in  20  dilution,  a  guarded 
opinion  is  given  and  the  case  stated  to  be  "  probably  enteric  fever  "  ;  if 
both  preparations  are  unchanged,  the  case  is  reported  as  "probably  not 
enteric  fever." 

In  the  measured  dilutions  it  may  be  said  that  if  in  half-an-hour  there 


484  APPENDIX 

is  a  positive  result  with  the  50  per  cent.,  5  per  cent.,  and  O5  per  cent., 
the  case  is  undoubtedly  one  of  typhoid  fever,  and  if  in  half-an-hour  there 
is  no  reaction  in  all  three,  the  result  is  definitely  negative.  Intervening 
degrees  of  reaction  must  each  be  judged  on  its  own  merits,  and  a 
subsequent  examination  made. 

From  the  compilation  of  a  large  number  of  cases,  the  New  York 
Health  Board  concludes  that  Widal's  reaction  is  present  in  typhoid 
fever : — 

From  the  fourth  to  seventh  day  in  70  per  cent,  of  the  cases. 

From  the  eighth  to  fourteenth  day  in  80  per  cent,  of  the  cases. 

During  the  third  and  fourth  weeks  in  90  per  cent  of  the  cases. 

It  is  absent  throughout  in  5  to  10  per  cent,  of  the  cases. 

Widal's  reaction  persists  in  the  blood  for  months,  or  even  years,  but 
after  three  or  four  months  is  usually  feeble. 

Differentiation  of  B.  Typhosus 

On  p.  48  will  be  found  some  of  the  chief  distinguishing  tests  for  the 
typhoid  bacillus,  which  produces  no  gas  in  any  media,  does  not  coagu- 
late milk,  and  stains  by  Gram's  method.  McConkey's  test  for  B.  coli 
may  also  be  used.  The  medium  which  he  makes  use  of  is  bile-salt- 
lactose  agar,  which  is  prepared  as  follows  :  To  1000  c.c.  of  tap- water  in 
a  flask  are  added  2  per  cent,  of  peptone,  0'5  per  cent,  of  sodium  tauro- 
cholate,  and  1*5  per  cent,  of  agar.  The  flask  is  autoclaved  at  105°  to 
110°  C.  for  one  and  a  half  hours.  The  mixture  is  then  cooled,  mixed 
with  white  of  egg,  and  filtered ;  then  1  per  cent,  of  lactose  is  added. 
The  medium  is  distributed  into  test-tubes,  10  c.c.  in  each,  which 
are  sterilised  by  steaming  for  15-20  minutes  on  each  of  three  successive 
days.  Plates  are  made  and  incubated  at  4*2°  C.  for  forty-eight  hours. 
There  is  a  marked  difference  between  the  colonies  of  the  organisms  of 
the  typhoid  group  and  those  of  the  colon  group.  Of  the  typhoid  group 
the  surface  colonies  are  small,  round,  raised,  and  semi-transparent,  the 
deep  one  lens-shaped,  white,  and  opaque,  the  medium  remaining  clear. 
Of  the  colon  group  the  surface  colonies  are  roundish  or  irregular,  with 
flattened  tops,  opaque,  white,  with  a  yellow  or  orange  spot  in  the  centre  ; 
a  few  have  a  haze  round  them.  The  deep  colonies  all  have  a  haze 
round  them,  and  are  lens-shaped  and  orange-white.  The  haze  is  due 
to  precipitation  of  the  sodium  taurocholate  by  acid  produced  by  fermen- 
tation of  the  lactose.  McConkey  and  Hill  *  have  further  modified  this 
method  by  the  use  of  a  bile-salt  broth,  composed  as  follows :  Sodium 
taurocholate,  0'5  per  cent. ;  glucose,  0*5  per  cent. ;  peptone,  2  per  cent.  ; 
water,  100  c.c.  The  constituents  are  dissolved  by  heat,  and  the  mixture 
is  filtered.  After  filtration,  sufficient  neutral  litmus  is  added  to  give  a 
distinct  colour,  and  the  medium  is  then  distributed  into  Durham's  fer- 
mentation tubes.  These  are  ordinary  test-tubes  containing  a  piece  of 
light-glass  tubing,  about  an  inch  in  length,  closed  at  the  upper  end. 

*  Thompson  Yates  Laboratories  Report,  1901,  vol.  iv.,  part  i.,  p.  151. 


APPENDIX  485 

This  acts  as  a  miniature  gas-holder  if  fermentation  of  the  medium 
occurs.  The  tubes  are  finally  steamed  for  twenty  minutes  for  each  of 
three  successive  days.  For  the  examination  of  water  1  c.c.  is  added  to 
each  tube,  and  several  are  inoculated  and  incubated  at  42°  C.  for  forty- 
eight  hours.  If  the  colon  bacillus  be  present,  the  medium  becomes 
uniformly  red,  and  is  permeated  with  small  gas  bubbles,  while  the  little 
tube  is  filled  with  gas.  Subsequently,  plates  may  be  made  from  the 
tubes  with  .the  bile-salt  agar  medium. 

Examination  of  Malarial  Blood 

1.  Fresh  Blood. — Thoroughly  clean  a  cover-glass  and  wash  a  finger  of 
the  patient.     Then  prick  the  finger  and  squeeze  out  a  drop  of  blood. 
This  first  drop  of  blood  should  be  rejected.     But  when  a  second  smaller 
drop  appears,  just  touch  its  surface  with  the  clean  cover-glass.     Now 
place  the  cover-glass  on  a  clean  slide,  but  do  not  exert  any  pressure 
upon  it.     Under  the  weight  of  the  cover-glass  the  blood  will  now  spread 
out  into  a  very  thin  film.     On  examination  under  the  microscope  or  by 
the  naked  eye  it  will  be  seen  that  the  blood  corpuscles  have,  roughly 
speaking,  assumed  the  following  zones  : — 

(a)  A  zone  of  scattered  corpuscles  immediately  surrounding  a  central 
portion  empty  of  corpuscles  and  devoid  of  colour.  This  "scattered" 
zone  is  composed  of  isolated,  compressed,  and  much  expanded 
corpuscles. 

(6)  Outside  this  first  zone  is  one  composed  of  corpuscles  just  touching 
each  other  by  the  margin.  This  has,  therefore,  been  called  the  single 
layer  zone. 

(c)  The  third  zone  lies  still  further  outside,  and  is  composed  of 
heaped-up  corpuscles,  overlapping  each  "other  and  often  in  rouleaux. 
Beyond  them  is  the  area  of  free  haemoglobin,  and  valuable  as  enabling 
the  observer  to  see  if  there  are  pigment  parasites  present  in  the  blood. 

The  ordinary  pigmented  amoeboid  forms  of  the  parasite  will  generally 
be  found  in  the  single  layer  zone,  whilst  the  flagellated  bodies,  if  present, 
will  be  seen  chiefly  in  zone  (c). 

Pigmented  leucocytes  may  appear  anywhere  in  the  field  of  the 
microscope. 

The  intra-corpuscular  parasites  may  generally  be  detected  because 
of  their  amoeboid  movements,  pigmentation,  feeble  definition,  and  effect 
upon  the  corpuscle  containing  them. 

2.  Stained  Preparation. — Whilst  it  is  always  best  to  examine  malarial 
blood  in  a  fresh  state  if  possible,  it  is  generally  desirable  to  make  more 
permanent  preparations.     This  may  be  done  as  follows : — Make  upon  a 
clean  slide  a  very  thin  film  of  the  malarial  blood  (by  drawing  a  needle 
or  edge  of  cigarette  paper  over  film).    Allow  it  to  dry  in  the  air.     Then 
wash  the  slide  containing  the  dried  film  with  weak  acetic  acid  (say  two 
or  three  drops  of  glacial    acetic  to  an    ounce  of  water)  to   clear   the 
haemoglobin.     This  may  also  be  accomplished  by  dropping  on  the  slide 
a  little  alcohol,  which  may  be  dried  up  in  several  minutes'  time  with 


486  APPENDIX 

filter-paper.  After  either  of  these  methods  has  been  adopted,  stain  the 
film  for  thirty  seconds  with  a  concentrated  aqueous  solution  of  methylene- 
blue  (or  the  following  solution  for  the  same  period  of  time :  Borax, 
5  parts ;  methyl  ene-blue,  2  parts ;  water,  100  parts).  Wash  in  water, 
dry  with  filter-paper,  and  mount  in  xylol-balsam  under  a  cover-glass.* 
Loffler's  blue  or  carbol  thionin  may  be  used.  For  double  staining, 
Jenner's,  Romanowsky's,  or  Leishman's  stains  may  be  used. 

To  demonstrate  flagella,  proceed  as  follows : — Take  a  piece  of  thick 
blotting-paper,  3  x  1 J  inches,  with  a  round  hole  in  the  middle  the  size 
of  an  ordinary  cover-glass.  Moisten  the  blotting-paper  and  place  it  on 
a  clean  slide.  Take  a  drop  of  the  blood  on  another  slide  which  has 
been  breathed  upon,  and  invert  it  on  the  blotting-paper  (moist  cell). 
In  thirty  minutes  separate  and  dry  the  blood-film  on  both  slides  by 
gentle  warming  over  the  lamp.  Fix  with  absolute  alcohol,  which  may 
be  allowed  to  evaporate  or  be  dried  with  filter-paper.  Wash  with 
acetic  acid  (15  per  cent.)  to  dissolve  out  the  haemoglobin,  wash  in  water, 
and  dry  as  before.  Stain  the  dried  film  with  carbol-fuchsin  (20  per  cent.) 
for  six  to  eight  hours.  Wash  and  mount  as  before. 

Bacteriological  Examination  of  Oysters 

Particular  attention  should  be  paid  to  the  (a)  washings  of  the  shell, 
(b)  the  liquor  in  the  pallial  cavity,  and  (c)  the  contents  of  the  alimentary 
canal  of  the  oyster.  The  two  latter  are  the  chief  parts  for  examination 
in  the  ordinary  course,  and  to  obtain  knowledge  of  the  contained 
bacteria  the  method  to  adopt  is  as  follows  : — 

Method. — Thoroughly  cleanse  the  oyster  shells  by  scrubbing  with 
soap  and  water,  rinse  under  the  tap,  and  again  in  sterile  water.  Also 
the  hands  of  the  bacteriologist  should  be  thoroughly  cleansed  and 
rinsed  in  antiseptic  (e.g.  1-1000  corrosive  sublimate)  and  sterile  water. 
Now  lay  the  oysters  on  the  table  with  the  flat  shell  uppermost,  and 
open  with  a  sterile  knife.  Pour  the  pallial  liquor  into  a  sterilised  flask 
or  capsule,  and  cut  up  the  body  of  the  oyster,  adding  the  pieces  to  the 
liquor  or  to  another  flask.  Add  to  the  flasks  of  liquor  and  of  oyster 
pieces,  or  to  the  one  flask  containing  both  sufficient  sterile  water  (100 
c.c.  or  1000  c.c.  as  desired).  The  emulsion  is  now  to  be  cultured  as 
follows,  adding  in  each  case  suitable  quantities  of  the  emulsion,  e.g.  (10 
c.c.  or  5  c.c.  or  1  c.c.  or  '5  c.c.)  : — Three  tubes  of  broth  (for  indol  forma- 
tion) ;  three  tubes  of  phenolated  broth  (for  B.  coli  and  its  allies,  and  also 
for  secondary  plate  cultivation) ;  three  tubes  of  M'Conkey  medium, 
bile-salt-glucose  peptone  (for  B.  coli  and  its  allies,  coloration  and  gas)  ; 
three  tubes  of  freshly  sterilised  milk,  heated  after  inoculation  to  80°  C. 
for  15  minutes,  and  cultured  anaerobically  (for  B.  enteritulis  sjwrogenes) ; 
three  tubes  of  litmus  milk  (for  acid  and  clotting) ;  three  gelatine 
"  shake "  cultures  (for  gas  production) ;  three  plates  of  phenolated 
gelatine  and  three  of  ordinary  gelatine ;  and  three  plates  of  agar  for 
incubation  at  37°  C.  For  quantitative  estimation  of  colonies  011  the 

*  See  also  Tropical  Diseases  (Manson),  p.  40. 


APPENDIX  487 

plates,  it  will,  of  course,  be  necessary  to  multiply  up  according  to  degree 
of  dilution  of  the  pallial  liquor  with  sterile  water  in  making  the  emulsion 
in  the  first  instance.* 

Examination  Of  Urine. — Urine  is  examined  in  the  same  way  as 
water  or  sewage  effluent.  Plates  (gelatine  and  agar)  and  sub-cultures 
are  made  in  the  usual  way.  The  urine  should  also  be  centrifugalised 
and  the  sediment  carefully  examined  by  microscope  and  culture,  and  if 
necessary  inoculated  into  guinea-pigs.  The  organisms  chiefly  to  be 
looked  for  are  B.  typhosus  (in  cases  of  typhoid  fever),  B.  tuberculosis, 
septic  organisms,  and  B.  coli. 

Examination  Of  Ice-cream. — Ice-cream  usually  contains  vast 
numbers  of  bacteria.  It  is  examined  in  the  same  way  as  milk,  and 
requires  high  dilution  before  examination. 

Examination  Of  Meat,  Fish,  etc. — Mince  a  portion  of  the  unsound 
meat  or  potted  meat  or  fish  by  aid  of  sterile  scissors  and  forceps,  and 
make  an  emulsion  in  broth  in  a  flask  at  42°  C.  (for  thirty  minutes). 
Shake.  Pipette  off  10  c.c.  of  extract  for  inoculation  of  animals.  Make 
plates  and  further  tube  cultures  of  the  emulsion.  Incubate  duplicates 
anaerobically  in  Bulloch's  apparatus.  Feed  animals  on  portions  of  the 
samples. 

Methods  of  Examination  of  Sewage  and  Sewage  Effluents 

The  sample  of  sewage  or  effluent  to  be  examined  must  be  collected 
in  the  same  manner  as  in  water. 

1.  Physical  Examination. — Take   note    of   quantity,  colour,  character 
and  amount  of  deposit  and  suspended    matter,  reaction,   temperature, 
bubbles  of  gas,  etc. 

2.  Dilution. — This  must  be  carried  out  as  in  the  examination  of  milk, 
500-1000  times. 

3.  Quantitative  Examination. — Make  plates  on   Petri  dishes,  gelatine 
for  incubation  at  20°  C.,  and  agar  at  37°  C.     Sewage  is  rich  in  intestinal 
germs,  most  of  which  grow  luxuriantly  at  blood-heat. 

4.  Qualitative   Examination. — The    three  chief  organisms   of  sewage 
are  :  (a)  B.  coli  (p.  46),  (6)  B.  enteritidis  sporogenes  (pp.  156  and  307),  and 
(c)  sewage  streptococcus  (p.   155).     It  is  necessary,  therefore,  to  examine 
particularly  for  these  organisms.     It  may  also  be  necessary  to  estimate 
quantitatively  for  B.  coli  and  B.  enteritidis  sporogenes.^ 

5.  Subsidiary  Differential  Tests. — Inoculation  of  animals  test ;  produc- 
tion of  gas  in  gelatine  "  shake  "  cultures  in  twenty-four  hours  at  20°  C. ; 
acid  clotting  of  litmus  milk  in  twenty-four  hours  at  37°  C. ;  greenish- 
yellow  fluorescence  in  neutral-red  broth  cultures  in  twenty-four  hours 
at  37°  C.  ;  the  production  of  indol  within  five  days  at  37°  C. ;  and  the 
bile-salt  broth  test  (growth,  gas,  and  acid). 

*  A  large  number  of  methods  and  modes  of  experiment  in  the  investigation  of 
Oysters  will  be  found  in  the  appendices  of  the  Fourth  Report  of  Royal  Commission 
on  Sewage  Disposal,  1904,  vol.  iii.,  pp.  191-309  (Houston). 

t  For  methods,  see  Royal  Commission  on  Sewage,  Second  Report,  1902,  p.  140 
(Houston). 


488  APPENDIX 

To  Clean  Glass  Apparatus 

Test-tubes  and  flasks  may  be  washed  in  a  bucket  with  hot  water  and 
soap  powder  or  soda,  or  boiled  in  the  same.  They  should  then  be 
cleaned  with  test-tube  brushes  and  inverted  for  draining.  Before  use 
they  must  be  sterilised.  Pipettes  may  be  treated  in  the  same  way,  and 
then  rinsed  through  with  rectified  spirit,  and  sterilised  in  the  hot-air 
oven.  When  'test-tubes  and  pipettes  are  infected,  they  should  be 
treated  in  a  similar  manner,  and  also  placed  in  strong  disinfectant  or 
nitric  acid  (5  per  cent.).  Greasy  slides  should  be  placed  in  alcohol  and 
acid  (5  per  cent.  HC1  or  H2SO4)  for  several  hours,  and  then  rinsed  in 
water.  Greasy  cover-slips  may  be  treated  in  the  same  way,  or  boiled 
in  chromic  acid  (10  per  cent.)  and  washed  in  acid  alcohol  and  water. 

Choice  of  Medium 

This  must  be  left  very  largely  to  individual  experience  and  the 
objects  of  the  investigation.  In  a  general  way  the  constituents  of  the 
various  media  described  indicate  the  purposes  to  be  obtained.  The 
general  standard  liquid  media  are  bouillon  and  milk,  the  solid  media  are 
gelatine  (for  room  temperature  cultivation)  and  agar  (for  blood-heat).  In 
tropical  countries  a  combination  of  the  two  may  be  used.  Further, 
just  as  gelatine  is  a  solid  bouillon,  so  gelatinised  milk  may  be  used  when 
a  solid  milk  medium  is  required.  For  anaerobes  glucose  and  formate 
media  are  commonly  used.  There  are,  of  course,  various  media  used  for 
different  species  of  organisms.  For  the  streptothrix  group  including  B. 
tuberculosis,  glycerine  media  and  potato  are  used.  To  isolate  the  B.  typhosus, 
carbolised  media  and  Eisner  are  taken.  Chromogenic  bacteria  nearly 
always  grow  well  on  potato.  The  use  of  litmus  milk,  beer  wort,  wort 
gelatine,  milk  agar,  etc.,  is  sufficiently  designated  in  the  names  of  the 
media. 

Preservation  of  Media. — Media  may  be  kept  in  good  condition  for 
months  if  a  few  simple  precautions  are  borne  in  mind.  The  tubes  or 
flasks  containing  the  medium  must  be  effectually  sealed,  either  with  caps, 
corks,  or  paraffin.  The  store  of  media  must  then  be  kept  in  a  closed 
metal  box,  and  in  a  cool  dark  place. 


INDEX 


ABSCESS  formation,  311 

bacteria  of,  312-313 
Acetous  fermentation,  102 
Acid-fast  bacteria,  358-369 

classification  of,  359 

of  human  origin,  360 

of  butter  and  milk,  861 

of  grass  and  manure,  364 

differential  diagnosis,  365 

streptothrix,  367 
Actinomycosis,  321 
Aerobic  organisms,  23 
Agar,'l6 
Air,  bacteriology  of,  76-91 

dust  and  bacteria,  76 

examination  of,  73-75 

moisture  and  bacteria,  79 

of  sewers,  82 

currents  and  bacteria,  84 

expired,  79-81 

of  workshops,  85 

bacteria  and  gravity,  83 

of  bakehouses,  86 

standard  of  bacteria  in,  79,  91 

of  railway  tubes,  88,  90 

pathogenic  bacteria  in,  91 

of  House  of  Commons,  88 

passages,  bacteria  in,  80 
Alcohol,  formation  of,  96 
Alcoholic  fermentation,  96 
Alexines,  412 

Alformant  lamp  for  disinfection,  443 
Algae  in  water,  35 
Ammoniacal  fermentation,  110 
Amylolytic  ferments,  95 
Anaerobic  organisms,  23 

methods  of  culture,  117-119 

in  hydrogen,  117 

in  glucose  agar,  118 

in  Franke!' s  tube,  118 

in  Buchner's  tube,  118 

4S9 


Andrewes  on  air  of  central  railway  tube, 

90 

Aniline  dyes,  455,  458 
Antagonism  of  organisms,  30 
Antibiosis,  29 
Anthrax,  315-319 

clinical  characters  of,  315 

pathology  of,  316 

spores  of,  316 

bacillus  of,  316 

in  sewage,  177 

channels  of  infection,  317 
Antiseptics,  433 

definition  of,  433 

some  of  the  chief,  439-444 
Antitoxins,  405 

preparation  of,  425 

use  of,  429 

unit  of,  428 

effect  of,  430 

Appendix  on  technique,  453 
Arthrospores,  12,  13 
Artificial  purification  of  water,  64-70 
Ascospores,  14,  98 
Asiatic  cholera,  384 
Association  of  organisms,  29 
Attenuation  of  virulence,  31 
Autoclave,  25 

BACILLUS,  definition  of,  88 
aceti,  102 

acidi  lactici,  105,  106,  196 
anthracis,  316 
aquatilis,  45 
botulinus,  269 
butyricus,  108-110 
capillareus,  155 
cloacae  fluorescens,  155 
colicommunis,  46-51,  56-60,  154,  466, 

477 
tests  for,  48-51,  466 

2   H   2 


490 


INDEX 


Bacillus — 

diphtheria,  288 
enteritidis  of  Gaertner,  269 
enteritidis  sporogenes,  45,   154,  156, 

307 

fluorescens  liquefaciens,  45 
fluorescens  non-liquefaciens,  45 
fluorescens  stercoralis,  155 
fusiformis,  155 

friburgensis,  Nos.  1  and  2,  363 
of  cholera,  385 
of  Binot,  364 
of  diarrhoea,  305 
of  dysentery,  403 
of  Grassberger,  364 
of  influenza,  321 
lactis  erythrogenes,  45,  200,  201 
lactis  pituitosi,  200 
lactis  viscosus,  200 
liquefaciens,  45 
mallei,  323 

membranous  patulus,  155 
of  leprosy,  398 
phlei,  364 

of  glanders  (mallei),  323 
mesentericus,  45 
of  Moeller,  362 
mycoides,  45 

or  malignant  oedema,  144 
No.  41,  245 
of  Rabinowitsch,  361 
of  symptomatic  anthrax,  142 
of  plague,  392 
prodiffiosus,  200 
pseudo-  tuberculosis,  358 
pyo-cyaneus,  157 
pyogenes  cloacinus,  155 
radicicola,  134 
saponacei,  200 
smegmatis,  360 
subtilis,  45 
subtilissimus,  155 
synxanthus,  201 
of  tetanus,  141,481 
of  tubercle,  327,  337 
typhosus,  48,  301 
of  yellow  fever,  400 

Bacteria,  action  of,  285 
composition  of,  9 

Bacteria,  in  sewage,  151-158 

and  wheat  supply,  131 

and  fixation  of  nitrogen,  131-139 

in  cheese-making,  241 

in  the  dairy,  178-251 

products  of,  406 

and  disease,  280 

the  higher,  6,  8 

in  soil,  116 
Bacterial  action,  285 


Bacterial  action — 

diseases  of  plants,  32 

treatment  of  sewage,  162-177 
Bacterio-purpurin,  10 
Bacteroids,  136 
Bakehouses,  bacteria  in,  86 
Ballard  on  soil  and  disease,  145 

on  epidemic  diarrhoea,  304,  308 
Beer  diseases,  110-113 
Berkefeld  filter,  71 
Beri-beri,  404 
Biogenesis,  2 
Biology  of  bacteria,  1 
Bitter  fermentation,  112 
Blood  serum,  16 
Blue  milk,  201 
Booker  on  bacteria  of  epidemic  diarrhoea, 

306 

Boracic  acid,  441 
Boyce    and    others    on    bacteriological 

examination  of  water,  470-473 
Bread,  bacteria  in,  276-279 

sour,  277 

mouldy,  278 

sticky,  278 

red,  279 
Broth,  16 

Brownian  movement,  11 
Bubonic  plague,  388-396 
Buchner's  tube,  118,  466,  478 
Butter  bacilli,  the,  361-364 
Butter,  bacteria  in,  241 

making,  242-246 

examination  of,  479 

bacterial  flavouring  of,  242 
Butyric  fermentation,  107 

CARBOL-FUCHSIN,  455,  459 

Carbol-gelatine,  16 

Carbolic  acid  as  a  germicide,  441 

Carbonic  acid  gas  and  bacteria,  85-91 

Caries,  dental,  80 

Carson's  dairy  farm,  232 

Cellulose,  10 

Chamber,  moist,  464 

Channels  of  infection  in  disease,  284 

abnormal,  251 
Cheese,  bacteria  in,  241 

making,  246 

examination  of,  480 

poisonous,  251 
Chemical  products  of  bacteria,  406 

substances  as  disinfectants,  439-444 


INDEX 


491 


Chemical  products  of  bacteria — 

and  bacteriological  examination  of 

water  compared,  55 
tests  for  nitrification,  128-132 
Chemiotaxis,  11 

Chloride  of  lime  as  a  germicide,  440 
Cholera,  384 

bacillus  of,  385 
diagnosis  of,  387 
and  nitration,  66  . 
and  milk,  223 
Chromogenic  bacteria,  406 
Clams  and  bacterial  infection,  266 
Clark's  process,  64 
Classification,  5 
Clowes  on  bacterial  treatment  of  sewage, 

164 

Coccus,  definition  of,  6 
Cockles  and  bacterial  infection,  263-266 
Collingridge  on  ice-cream  poisoning,  274 
Colon  bacillus,  see  B.  coli  communis,  46 
Comma  bacillus,  385 
Commensalism,  133 

Commissions  on  food  preservatives,  230 
leprosy,  399 
plague,  394,  425 
sewage  disposal,  49,  157,  161,  162, 

172,  177,  261,  262,  469,  485 
tuberculosis  (1898)  270,  (1895)  339, 

343,  (1901)  345 
vaccination,  417 
Composition  of  bacteria,  9 
Conditions  affecting  bacteria  in  water, 

60 

Contact  beds  for  sewage,  169 
Contagion,  284 

Contamination,  organisms  of,  56 
Corrosive     sublimate     as     disinfectant, 

440 

Counter  (Wolfhugel),  465 
Cover-glass  preparations,  455 
Cream,  bacteria  in,  240 
Crenothrix  polyspora,  36 
Cresol  as  a  germicide,  441 
Cultivation  beds,  169 
Culture  media,  16 
Cultures,  anaerobic,  117 
hanging  drop,  455 
plate,  453 
pure,  456 
shake,  467 
sub-culture  of,  456 

DECOMPOSITION  bacteria,  123 


Delepine  on  bacteria  in  milk,  192-194 

Denitrifying  bacteria,  123 

Dental  caries,  80 

Deodorants,  433 

Desiccation,  18 

Diagnosis,  463 

Diarrhrea  of  infants,  304 

and  milk,  223 

conditions  favourable  to,  304,  308 

and  soil,  308 

bacteria  of,  305 
Diphtheria,  287-296 

antitoxin  of,  425-431 

bacillus  of,  288 

bacillus  in  throat,  293 

toxins  of,  426 

prevention  of,  294 

and  milk  supply,  211 

and  school  influence,  292 

pseudo-bacillus  of,  295 

diagnosis  of,  290,  481 

Diplococcus,  definition  of,  7 
of  gonorrhoea,  314 
in  pneumonia,  320 

Directions  for  estimating  disinfectants, 

etc.,  434 

Disease,  production  of,  280-287 
Diseases  of  beer,  110-113 

of  plants,  32 

conveyed  by  water,  53 

and  soil,  145 
Disinfectants,  439 
Disinfection,  432-451 

means  of,  435 

by  heat,  436 

by  chemicals,  439-444 

of  a  room,  444 

of  walls,  445 

of  bedding,  445 

of  garments,  445 

of  excreta,  445 

of  wounds,  445 

of  hands,  446 

of  books,  etc. ,  446 

of  stables,  vans,  etc.,  446 

after  phthisis,  447 

after  small-pox,  449 

after  scarlet  fever,  449 

after  diphtheria,  450 

after  typhoid,  450 

after  cholera,  450 

after  plague,  450 

standards  of,  434 

Domestic  purification  of  water,  70 
Doriga  on  rats  and  plague  infection,  390 
Dunham's  solution,  388 


492 


INDEX 


Dysentery,  404 

EARTH  temperatures  and  disease,   145, 

308 

Effluents,  158,  175 
Eisner's  medium,  467 
Endospores,  12,  14 
Enteric  fever  (see  typhoid),  298-304 
Enzymes,  94 
Equifex  disinfector,  438 
Examination ,  bacteriological — technique 
of,  453-463 

air,  73 

cholera,  387 

diphtheria,  481 

fish,  485 

ice-cream,  485 

leprosy,  398 

malarial  blood,  485 

meat,  485 

milk,  473-481 

oysters,  484 

sewage,  485 

soil,  117 

tetanus,  481 

typhoid,  482 

tubercle,  482 

urine,  485 

water,  463-473 

yeasts,  97 

Extracellular  poisons,  408 
External  conditions,  effect  of,  on  bac- 
teria, 15 

FERMENTATION,  92-115 

kinds  of,  94 

acetous,  102 

alcoholic,  96-102,  198 

ammoniacal,  110 

butyric,  107,  197 

lactic  acid,  104,  196 
Ferments,  organised,  94 

unorganised,  94 

chromogenic,  200 

curdling,  104,  197 

bitter,  112-199 

slimy,  199 

soapy, 200 
Films,  100 
Filters,  domestic,  71 

sterilisation  of,  72 
Filtration  of  milk,  228-230 

method  of  air-examination,  74 
Filtration  of  water,  65-72 
Filter-beds,  65 

Firth     and     Horrocks     on    pathogenic 
bacteria  in  soil,  148 


Fission,  12 

Fixing  specimens,  475 

Flagella,  11 

staining,  461 

Food,  bacteria  in,  178,  253 
Formaldehyde  and  formalin,  443 
Forms  of  bacteria,  6 
Foulerton  on  pollution  of  water,  63 

on  bacteria  in  oysters,  260 

on  streptothrix  group,  367 
Fowler  on  bacterial  treatment  of  sewage, 

174 

Fractional  sterilisation,  24 
Frankel's  pneumococcus,  320 
Frankland  on  bacteria  in  water,  37 

on  filtration  of  water,  65 
Freezing,  effect  of,  on  bacteria,  18 
Friedlander's  pneumo-bacillus,  320 

GAS,  production  of,  406 
Gathering-ground,  34 
Gelatine,  16 

carbol,  466 

liquefaction  of,  457 
Gemmation,  98 
Gentian-violet,  aniline,  455 
Germicidal  temperatures,  23-25 
Germicides,  439-444 
Gilbert  on  nitrification,  134 
Ginger-beer  plant,  137 
Glanders,  323 
Gonorrhcea,  314 
Gram's  method  of  staining,  458 

Nicolle's  modification  of,  459 
Gravity,  influence  on  bacteria,  83 
Gypsum  block,  98 

tLEMOCYTOZOA,  372 

Hsemamceba,  372 

Haldane  on   ventilation   of  workshops, 

85 

Hanging-drop  cultivations,  455 
Hansen's  method  of  dilution,  99 
Heat  as  steriliser,  23-25 
Heredity,  284 

Hesse's  method  of  air  examination,  74 
Hewlett  and   others  on  bacteriological 

examination  of  water,  470-473 
High  yeasts,  101 
Higher  bacteria,  6,  8 
Horrocks  and  Firth  on  soil  and  disease, 

148 


INDEX 


493 


Horrocks     on    classification     of    water 
bacteria,  44 

Hot-air  steriliser,  24,  25 

Houston  on  streptococci  in  water,  51 
on  pathogenic  bacteria  in  soil,  148 
on  sewage  bacteria,  153, 157,175, 177 
on  bacteria  in  oysters,  261 

Hydrogen  cultivation,  23 

Hydrophobia,  treatment  of,  419 

ICE,  bacteria  in,  274 
Ice-cream,  bacteria  in,  272-274 

manufacture  of,  272 

examination  of,  485 
Immunity,  405-431 

acquired,  413 

active,  412 

Ehrlich's  side  chain  theory,  414 

artificial,  412 

general  principles,  405-412 

natural,  413 

passive,  413 

theories  of,  413 

in  small-pox,  415 

in  rabies,  419 

in  typhoid,  424 

in  plague,  424 

in  diphtheria,  425 

in  cholera,  423 
Incubators,  17 
Indol,  formation  of,  467 

testing  for,  467 

Industries  and  bacteria,  113-115 
Infection,  channels  of,  284 
Influenza,  321 
Inoculation,  456 

Interpretations  of  bacteriology,  55 
Intracellular  poisons,  31 
Inversive  ferments,  95 
Involution  forms,  9 

JORDAN'S  classification  of  water  bacteria, 
44 

KEPHIR,  136,  198 

Kipp's  apparatus  for  producing  hydrogen, 
23 

Klebs-Loffler  bacillus,  288 

Klein  on  bacteria  in  oysters,  259 
on  bacteria  in  cockles,  263 
onbacillusenteritidis  sporogenes,  156, 
307 

Koch's  plate  method,  453 
steam  steriliser,  24,  25 
tubercle  bacillus,  327  et  seq. 
postulates,  281 


Koch  on  filtration  of  water,  66 

on  inter-communicability  of  tuber- 
culosis, 339  et  seq. 
comma  bacillus,  385 
bacillus  of  tubercle,  327,  337 
views  on  tuberculosis,  338-346 

Koumiss,  198 

LACTIC  acid  fermentation,  104,  196 
Leguminosae,  fixation  of  nitrogen  by,  131 
Leprosy,  396-400 

history  of,  396 

forms  of,  397 

bacillus  of,  398 

Light,  influence  upon  bacteria,  18-22 
Lingner's  apparatus  for  disinfection,  443 
Liquefaction  of  gelatine,  457 
Liquid  hydrogen  and  bacteria,  18 
Lloyd  on  Cheddar  cheese-making,  249 
Low  yeasts,  101 
Lymph,  glycerinated  calf,  416 
Lyon's,  Washington,  disinfector,  437 

MACERATION  industries,  113 
Malaria,  371-384 

kinds  of,  373,  375 

cycle  of  Golgi,  380 

parasites  in,  372 

microgametocytes  of,  375 

macrogametocytes  of,  376 

and  mosquitoes,  376 

anopheles  of,  377 

culex  and,  378 

examination  of  blood,  485 

preventive  measures,  382-384 

mosquito  breeding,  382 

destruction  of  mosquitoes,  383 

bites  of  mosquitoes,  383 

quinine,  384 
Malignant  oedema,  144 

bacillus  of,  144 
Mallein,  324 
Malta  fever,  402 

Manchester  sewage  treatment,  170-174 
Manson  on  malaria,  375  et  seq. 

on  plague,  390 
Martin  on  soil  and  disease,  147 

on  tuberculosis,  341-342 
Mastitis,  184 

M'Conkey's  bile-salt  method,  467,  484 
Meat  and  bacterial  infection,  267-272 

examination  of,  485 

poisoning  bacteria,  269 

tuberculous,  270 

decomposed,  270 
Media,  culture,  16 


494 


INDEX 


Merismopedia,  8 
Metabiosis,  29 
Metachromatic  granules,  10 
Metchnikoff  on  phagocytosis,  414 
Methods  of  examination,  453 

Micrococcus,  definition  of,  6 

aquatilis,  Freud?,nreichiii  199 
gonorrhoea,  314 
tetrayonus,  313 
viscosus,  199 

Milk,  bacteriology  of,  178-251 
composition  of,  195 
incubation  period  for  bacteria  in,  179 
sources  of  pollution,  181-184 
number  of  bacteria  in,  184-194 
influence  of  time  and  temperature 

upon  bacteria  in,  185-194 
fermentation  bacteria  in,  196-202 
kinds  of  bacteria  in,  194 
disease-producing  power  of,  202 
lactic  acid  fermentation  of,  196 
butyric  fermentation  of,  197 
coagulation  fermentation  of,  197 
alcoholic  fermentation  of,  1 98 
anomalous  fermentation  of,  199 
and  tuberculosis,  203-207 
and  typhoid,  207-211 
and  cholera,  223 
and  epidemic  diarrhrea,  223-226 
and  diphtheria,  211-214 
and  scarlet  fever,  214-217 
and  sore-throat  illnesses,  219-223 
and  thrush,  218 

character  of  milk-borne  disease,  218 
prevention    of   milk-borne   disease, 

226 

method  of  protection,  227 
control  of  milk  supply,  227-240 
filtration  of,  230 
refrigeration  of,  228 
straining  of,  228 
sterilisation  of,  231 
of  Liverpool,  205 
pasteurisation  of,  231 
results  of,  235 
summary  of  control,  236 
products,  bacteria  in,  240-251 
examination  of,  473-481 
specialised  milks,  237 
and  economic  bacteria,  240-249 
and  municipal  depots,  237 
chromogenic  fermentation  of,  200 

Miquel's  method  of  air  examination,  74 

Modes  of  bacterial  action,  25 

Mohler  on  tuberculosis  of  the  udder,  203 

Moist  chamber,  464 

Moisture  necessary  for  bacteria,  18 


Morphology  of  bacteria,  6 
Motility,  11 

Mosquitoes  and  malaria,  376 
Mycoderma  aceti,  103 
Mycoprotein,  9 

NASAL  passages,  bacteria  in,  80 

Natural  purification  of  water,  60-64 

Needles,  platinum,  17 

New  soil  science,  138 

Newsholme  on  conditions  favourable  to 

diphtheria,  291 

on  causation  of  epidemic  diarrho?a, 
309 

on  disinfection  after  phthisis,  447 
Nitric  organism,  128 
Nitrification,  125-131 

chemistry  of,  125 

stages  in,  129 

bacteria  of,  129 
Nitrifying  organisms,  cultivation  of,  127, 

128 

Nitrogen,  fixation  of,  131-139 
Nitrogen-fixing  bacteria,  131 
Nitrous  organism,  126 
Niven  on  disinfection  after  phthisis,  447 
Nodules  on  roots,  bacteria  in,  133 

OCEAN  bacteria,  36 

Oidium  albicans,  218 

Oxygen  necessary  for  bacteria,  23 

Oysters  and  typhoid  fever,  253-263 

poisoning,  symptoms  of,  256 

infection,  257 

and  disease,  prevention  of,  262 

examination  of,  484 

FAKES'  formate  broth  method,  467 
Paraform  for  disinfection,  443 
Parasitism,  25 
Parietti's  method,  466 
Pasteur  on  fermentation,  93 
Pasteur's  treatment  of  rabies,  419 
Pasteur  filter,  71 
Pasteurisation  of  milk,  231-236 
Pathogenic  bacteria,  in  soil,  140 

in  water,  53 
Perlsucht,  333 
Petri  dishes,  453 
Phagocytosis,  413-414 
Phosphorescence,  22 
Pigment,  formation  of,  406 
Place  of  bacteria  in  nature,  4 


INDEX 


495 


Plague,  388-396 

varieties  of,  388 

symptoms  of,  388 

and  rats,  390 

distribution  of,  389 

bacillus  of,  392 

administrative  control  of,  394 

vaccination  for,  424 

diagnosis  of,  396 
Plant  diseases,  32 
Plasmolysis,  9 
Plate  cultures,  453 
Platinum  needles,  17 
Pleomorphism,  9 
Pneumonia,  319 

bacteria  of,  320 
Pneumo-bacillus,  320 
Pneumococcus,  320 
Polymorphism,  9 
Postulates,  Koch's,  281 
Potato  medium,  16 
Pouchet's  aeroscope,  74 
Power  on  milk-borne  scarlet  fever,  214 
Products  of  bacteria,  406 
Proteolytic  ferments,  95 
Proteus  family,  154,  45 

cloacinus,  154 

vulgaris,  154 

Zenkerl,  154 

mirabilis,  154 

Pseudo-diphtheria  bacillus,  295     . 
Pseudo-tuberculosis,  355-358 
Purification  of  water,  60-72 

natural,  60-64 

domestic,  70 

artificial,  64-70 
Pus,  311 
Pyocyanin,  313 
Pyoxanthose,  313 

QUANTITATIVE  standard  of  water  bacteria, 
42 

air  bacteria,  77 

milk  bacteria,  191 

soil  bacteria,  116 
Quarter-evil,  142 

clinical  characters,  143 

RAHIKS,  treatment  of,  419 

forms  of,  420 

pathology  of,  420 

results  of  treatment,  423 
Red  milk,  200 

Reproduction  of  bacteria,  modes  of,  11 
Relnmc,  113 


Rivers,  natural  purification  of,  38,  60-64 
Robertson  on  soil  and  typhoid,  147 
Ross  on  malaria,  380 
Rotch's  specialised  milk,  239 

Russel  on  butter-making,  244 
on  cheese-making,  247 

SACCHAROMYCETES,  biology  of,  97 
methods  of  examination,  99 
anomalous,  99 
apiculatus,  102 
aquifolii,  102 
cerevisice,  101 
conylomeratus,  102 
ellipsoideus  L,  101,  102 
ellipsoideus  II. ,  102 
exiyuus,  102 
Hansenii,  102 
illifiis,  102 
Ludwiffii,  99 
my  coder  ma,  102 
pastorianus  L,  102 
pastorianus  12.,  102 
pastorianus,  111. ,  1 02 
pyriformis,  102 
Sand  filtration  of  water,  65 
Saprophytes,  25 
Sarcina,  8 

Savage  on  bacteria  of  made  soil,  149 
Scarlet  fever,  296 
milk  and,  214 
bacteria  of,  296 
streptococcus  in,  297 
Sedgwick's  method  of  air  analysis,  75 
Seed  and  soil,  26 
Sedimentation,  62,  64 
Septic  processes,  311 

tank,  165-169,  174 
Sewage,  organisms  in,  151-158 
bacterial  treatment  of,  162-177 
constitution  of,  151 
examination  of,  154,  485 
organic  matter  in,  152 
inorganic  matter  in,  152 
number  of  bacteria  in,  153 
kinds  of  bacteria  in,  154 
spores  in,  153 
streptococcus  of,  155 
pathogenic  bacteria  in,  155 
nitrification   and  denitrification   in, 

157,  158,  166 
aerobic  and   anaerobic  bacteria  in, 

157,  158,  166 
disposal  of,  159 
chemical  treatment  of,  160 
biological  treatment  of,  158,  160-177 


496 


INDEX 


Sewage — 

irrigation  of,  161 

intermittent  filtration,  160 

effluents  and  pathogenic  organisms, 
175 

London  treatment  of,  164 

Manchester  treatment  of,  170 

Sutton  treatment  of,  169 

Exeter  treatment  of,  165 

septic  tank  method  of  treating,  165- 
169 

contact  bacteria  beds  method,  169 

Leicester  treatment  of,  173 

effect    of    bacterial    treatment    of, 

175 
Sewer  air,  82 

and  toxicity  of  bacteria,  83 
Shake  cultures,  467 
Shell-fish  and  bacteria,  253-266 
Sleeping  sickness,  403 
Small-pox,  416 
Smith,    Graham,    on    air  of  House  of 

Commons,  88 

Smith,  Horton,  on  typhoid  urine,  300 
Soil,  bacteriology  of,  116-150 

bacteria  in,  116 

composition  of,  119-122 

denitrification  in,  123 

examination  of,  117-119 

and  typhoid  fever,  146-148 

and  tetanus,  140 

polluted,  149 

kinds  of  bacteria  in,  119 

nitrification  in,  125-131 

nitrogen-fixing  bacteria  in,  131-139 

and  its  relation  to  disease,  145 

pathogenic  bacteria  in,  140 

classification  of  bacteria  from,  119, 
120 

symbiosis  in,  132,  136 
Sorensen's  dairy  farm  at  York,  228 
Species  of  bacteria,  28 
Specificity  of  bacteria,  28 
Spirillum,  definition  of,  88 

of  cholera,  385 

of  Obermeier,  8 

Spontaneous  generation,  3 
Sponges,  114 
Spores,  kinds  of,  12 

resistance  of,  12-15 

staining  of,  462 

of  yeasts,  98-99 
Staining  methods,  455-463 
Standard  of  sterilisation,  24 
Staphylococcus,  8,  311 

cereus  a  thus,  311 


Staphylococcus — 

pyogenes  aureus,  312 

pyogenes  albus  and  cHrens*  311 
Steatolytic  ferments,  95 
Steam,  as  a  disinfector,  436-438 

disinfectors,  437 

steriliser,  24 

saturated,  436 

superheated,  436 

current,  437 
Sterilisation,  23-25 

methods  of,  23-25 
Streptococcus,  7 

in  milk,  297 

in  water,  51 

in  sewage,  155 

of  scarlet  fever,  297 

pyogenes,  312 

conglomerate ,  297 

Hollandicus,  199 

Streptothrix  group,  367 

actinomycesi  321 

hominis,  368 

ringer,  etc. ,  369 

/a,  367 
Structure  of  bacteria,  6 
Sub-cultures,  456 

Sulphurous  acid  as  a  germicide,  441 
Suppuration,  311 
Swine  fever,  272 

Swithinbank  and  author  on  milk,  188-190 
Symbiosis,  29,  132,  136 
Symptomatic  anthrax,  142 

TABLE  of  economic  bacteria  in  soil,  120 
Temperature,  influence  of,  on  bacteria, 

17 
Tetanus,  140-142 

toxin  of,  141 

bacillus  of,  141,  481 
Thermophilic  bacteria,  17 
Thresh's  disinfector,  438 
Thresh  on  bacteriological  examination  of 

water,  469 
Thrush,  218 
Tobacco-curing,  114 
Toxins,  406-412 
Tropical  diseases,  370-404 
Tuberculin,  347 
Tuberculosis,  325-358 

pathology  of,  325 

varieties  of,  326 

history  of,  326 

conveyed  by  the  air,  79,  331 

and  the  milk  supply,  335 


INDEX 


497 


Tuberculosis — 

of  the  udder,  334 

giant  cells  in,  330 

bacillus  of,  327,  337 

bovine,  333,  337 

diagnosis  of  bovine,  346 

cultivation  of  bacillus  of,  328 

spores  of,  329 

relation  of  bacillus  to  disease,  330 

temperature  for  growth,  329 

toxins,  333 

of  horse  and  dog,  350 

of  cold-blooded  animals,  351 

of  animals,  349-351 

of  pig,  349 

of  birds,  350 

of  sheep,  349 

prevention  of,  352-357 

disinfection  in  cases  of,  447 

decline  of,  352 

and  overcrowding,  355 

channels  of  infection  in,  331 

inter-communicability  of,  338-346 

and  house  influence,  356 

pseudo-,  355-358 

Typhoid  fever,  298-304 
bacillus  of,  301 
effect  of  light  on  bacillus,  20 
pathology,  298 

bacillus  compared  with  B.  coli,  48 
bacillus  in  sewage,  175 
bacillus  in  drinking-water,  303 
tests  for  bacillus  of,  48,  468,  482,  484 
and  soil,  145 

conveyed  by  the  air,  78,  81 
and  milk  supply,  207-211 

Tyrotoxicon,  251 

UDDER  tuberculosis,  203,  334 
Unit  of  antitoxin,  428 
Urea,  120 

VACCINATION,  415-419 

effect  of,  417 
Vaccines,  415-425 

plague,  424 


Vaccines — 

cholera,  423 

small-pox,  415 
Vaccinia,  415 
Vacuolation,  9 
Variolation,  415 
Virulence,  attenuation  of,  31 

WARRINGTON  on  nitrification,  123  et  seq. 

Washington  Lyon  disinfector,  437 

Water,  bacteria,  classification  of,  44 
bacteria  in,  33,  44 
collection  of  samples,  33 
number  of  bacteria  in,  34,  36,  42, 

56 

examination  of,  463-473 
organisms  of  contamination,  56 
pathogenic  organisms  in,  53,  56 
multiplication  of  bacteria  in,  34 
natural  purification  of,  60-64 
river,  bacteria  in,  36-42 
artificial  purification  of,  64-70 
B.  coli  in,  46-60 
filtration  of,  65 
ordinary  bacteria  in,  45 
domestic  purification  of,  70 
London,  bacteria  in,  38-42 
quantitative  standard  in,  42 
quality  of,  44-60 
sewage  bacteria  in,  45 
pollution  of,  54-60 
sea,  bacteria  in,  36 

Watercress,  bacteria  of,  279 

Wheat  supply  and  bacteria,  131 

Widal  reaction,  482 

Wolfhiigel's  counter,  465 

Wool-sorters'  disease,  318 

YEASTS,  14,  97-102 
Yellow  fever,  400-402 

bacteria  in,  400 

and  mosquitoes,  402 
Yellow  milk,  201 

ZIEHL-NEELSEN  stain,  459 


PRINTED   BY 

OLIVER  AND  BOYD 

EDINBURGH 


